Mouse cytokine receptor

ABSTRACT

Cytokines and their receptors have proven usefulness in both basic research, animal models, and as therapeutics. The present invention provides a new cytokine receptor designated as “mouse Zcytor16,” which can bind and antagonize the IL-TIF cytokine.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/090,365, filed Mar. 4, 2002, which claims benefit of ProvisionalApplication 60/273,035, filed on Mar. 2, 2001, and ProvisionalApplication 60/279,232, filed on Mar. 27, 2001, all of which areincorporated herein by reference. Under 35 U.S.C. § 119(e)(1), thisapplication claims benefit of said Provisional Applications.

TECHNICAL FIELD

The present invention relates generally to a new protein expressed bymouse cells that is a murine ortholog to human Zcytor16 (U.S. patentapplication Ser. No. 09/728,911). In particular, the present inventionrelates to a novel gene that encodes a receptor, designated as “mouseZcytor16,” and to nucleic acid molecules encoding mouse Zcytor16polypeptides, and antibodies to the polypeptide.

BACKGROUND OF THE INVENTION

Cytokines are soluble, small proteins that mediate a variety ofbiological effects, including the regulation of the growth anddifferentiation of many cell types (see, for example, Arai et al., Annu.Rev. Biochem. 59:783 (1990); Mosmann, Curr. Opin. Immunol. 3:311 (1991);Paul and Seder, Cell 76:241 (1994)). Proteins that constitute thecytokine group include interleukins, interferons, colony stimulatingfactors, tumor necrosis factors, and other regulatory molecules. Forexample, human interleukin-17 is a cytokine which stimulates theexpression of interleukin-6, intracellular adhesion molecule 1,interleukin-8, granulocyte macrophage colony-stimulating factor, andprostaglandin E2 expression, and plays a role in the preferentialmaturation of CD34+ hematopoietic precursors into neutrophils (Yao etal., J. Immunol. 155:5483 (1995); Fossiez et al., J. Exp. Med. 183:2593(1996)).

Receptors that bind cytokines are typically composed of one or moreintegral membrane proteins that bind the cytokine with high affinity andtransduce this binding event to the cell through the cytoplasmicportions of the certain receptor subunits. Cytokine receptors have beengrouped into several classes on the basis of similarities in theirextracellular ligand binding domains. For example, the receptor chainsresponsible for binding and/or transducing the effect of interferons aremembers of the class II cytokine receptor family, based upon acharacteristic about 200 residue extracellular domain.

The demonstrated in vivo activities of cytokines and their receptorsillustrate the clinical potential of, and need for, other cytokines,cytokine receptors, cytokine agonists, and cytokine antagonists.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an alignment of human Zcytor16 (hZcytor16) (SEQ ID NO:2), andmouse Zcytor16 (mZcytor16) (SEQ ID NO:38). The “:” in the figureindicates amino acids that are identical between the mouse and humansequences, and the “.” in the figure indicates amino acids that areconserved substitutions. There is a 66.2% identity between the human andmouse sequences over the entire sequence (231 amino acid overlap).

BRIEF SUMMARY OF THE INVENTION

The present invention provides a novel receptor, designated “mouseZcytor16.” The present invention also provides mouse Zcytor16polypeptides and mouse Zcytor16 fusion proteins, as well as nucleic acidmolecules encoding such polypeptides and proteins, and methods for usingthese nucleic acid molecules and amino acid sequences.

DETAILED DESCRIPTION OF THE INVENTION

1. Overview

An illustrative nucleotide sequence that encodes mouse Zcytor16 isprovided by SEQ ID NO:37. The encoded polypeptide has the followingamino acid sequence: MMPKHCLLGL LIILLSSATE IQPARVSLTL QKVRFQSRNFHNILHWQAGS SLPSNNSIYF VQYKMYGQSQ WEDKVDCWGT TALFCDLTNE TLDPYELYYGRVMTACAGRH SAWTRTPRFT PWWETKLDPP VVTITRVNAS LRVLLRPPEL PNRNQSGKNASMETYYGLVY RVFTINNSLE KEQKAYEGTQ RAVEIEGLIP HSSYCVVAEM YQPMFDRRSPRSKERCVQIP (SEQ ID NO:38). The 230 amino acid polypeptide represents theextracellular domain, also called a cytokine-binding domain, of a newclass II cytokine receptor. Features of the Zcytor16 polypeptide includeputative signal sequences at amino acid residues 1 to 23 of SEQ IDNO:38, and a mature soluble receptor polyeptpide from residues 24 to 230of SEQ ID NO:38. The receptor has two fibronectin III domains, alsocalled immunoglobulin superfamily (Ig) domains, characteristic of theclass II cytokine receptor family that comprise amino acid residues 31to 122 (fibronectin III domain I), and 131 to 229 (fibronectin IIIdomain II) of SEQ ID NO:38, and a linker that resides between the Igdomains (i.e., at amino acid residues 127-130 of SEQ ID NO:38). Thusmolecules of the present invention include polypepetides that include acytokine binding domain comprising amino acids 31 to 229 of SEQ IDNO:38. Moreover, additional variants of the Zcytor16 polypeptide includepolyepeptides that comprise amino acid residues 27 to 122, 24 to 122, 31to 126, 27 to 126, or 24 to 126 (fibronectin III domain I), and 131 to229 or 230 (fibronectin III domain II) of SEQ ID NO:38, and a linkerthat resides between the Ig domains (i.e., at amino acid residues123-130, or 127-130 of SEQ ID NO:38). Thus molecules of the presentinvention include polypepetides that include a cytokine binding domaincomprising amino acids 24, or 27, to 229 or 230 of SEQ ID NO:38. Inaddition, zcytor16 contains conserved motifs and residues characteristicof class II cytokines: an SXWS (SEQ ID NO:46)-like motif from residue219-222 of SEQ ID NO:38; conserved Tryptophan residues at residues 46,71, and 113 of SEQ ID NO:38; and conserved Cysteine residues at residues77, 85, 205, and 226 of SEQ ID NO:38. The mouse Zcytor16 gene isspecifically expressed in Lung, Pancreas, Placenta, Salivary Gland,Skeletal Muscle, Skin, Small Intestine, Smooth Muscle, Spleen, Stomach,and Testis cDNAs and is expected to be expressed in specific mononuclearcells, such as activated T-cell and B-cell subsets. Alignment of thepolypeptide sequences shows that mouse Zcytor16 (SEQ ID NO:38) is anortholog of the human Zcytor16 sequence (SEQ ID NO:2) (U.S. patentapplication Ser. No. 09/728,911) as shown in FIG. 1.

Another illustrative nucleotide sequence that encodes a preferred mouseZcytor16 is provided by SEQ ID NO:47. The encoded polypeptide has thefollowing amino acid sequence: MMPKHCLLGL LIILLSSATE IQPARVSLTPQKVRFQSRNFH NILHWQAGSSLPSNNSIYFVQYKMYGQSQWEDKVDCWGTTALFCDLTNETLDPYELYYGRVMTACAGRHSAWTRTPRFFPWWETKLDPPVVTITRVNASLRVLLRPPELPNRNQSGKNASMETYYGLVYRVFIINNSLEKEQKAYEGTQRAVEIEGLIPHSSYCVVAEMYQPMFDRRSPRSKERCVHWP (SEQ ID NO:48). The 230 amino acidpolypeptide represents the extracellular domain, also called acytokine-binding domain, of a new class II cytokine receptor. The SEQ IDNO:48 sequence differs from SEQ ID NO:38 by two amino acids: amino acid30 (Leu) and 228 (Gln) shown in SEQ ID NO:38 are amino acids 30 (Pro)and 228 (His) shown in SEQ ID NO:48. Features of the Zcytor16polypeptide include putative signal sequences at amino acid residues 1to 23 of SEQ. ID NO:48, and a mature soluble receptor polyeptpide fromresidues 24 to 230 of SEQ ID NO:48. The receptor has two fibronectin IIIdomains, also called immunoglobulin superfamily (Ig) domains,characteristic of the class II cytokine receptor family that compriseamino acid residues 31 to 122 (fibronectin III domain I), and 131 to 229(fibronectin III domain II) of SEQ ID NO:48, and a linker that residesbetween the Ig domains (i.e., at amino acid residues 127-130 of SEQ IDNO:48). Thus molecules of the present invention include polypepetidesthat include a cytokine binding domain comprising amino acids 31 to 229of SEQ ID NO:48. Moreover, additional variants of the zcytor16polypeptide include polyepeptides that comprise amino acid residues 27to 122, 24 to 122, 31 to 126, 27 to 126, or 24 to 126, (fibronectin IIIdomain I), and 131 to 229 or 230 (fibronectin III domain II) of SEQ IDNO:48, and a linker that resides between the Ig domains (i.e., at aminoacid residues 123-130, or 127-130 of SEQ ID NO:48). Thus molecules ofthe present invention include polypepetides that include a cytokinebinding domain comprising amino acids 24, or 27, to 229 or 230 of SEQ IDNO:48. In addition, zcytor16 contains conserved motifs and residuescharacteristic of class II cytokines: an SXWS (SEQ ID NO:46) -like motiffrom residue 219-222 of SEQ ID NO:48; conserved Tryptophan residues atresidues 46, 71, and 113 of SEQ ID NO:48; and conserved Cysteineresidues at residues 77, 85, 205, and 226 of SEQ ID NO:48.

An illustrative nucleotide sequence that encodes human Zcytor16 isprovided by SEQ ID NO:1. The encoded polypeptide has the following aminoacid sequence: MMPKHCFLGF LISFFLTGVA GTQSTHESLK PQRVQFQSRN FHNILQWQPGRALTGNSSVY FVQYKIYGQR QWKNKEDCWG TQELSCDLTS ETSDIQEPYY GRVRAASAGSYSEWSMTPRF TPWWETKIDP PVMNITQVNG SLLVWHAPN LPYRYQKEKN VSIEDYYELLYRVFINNSL EKEQKVYEGA HRAVEIEALT PHSSYCVVAE IYQPMLDRRS QRSEERCVEI P (SEQID NO:2). The 231 amino acid polypeptide represents the extracellulardomain, also called a cytokine-binding domain, of a new class IIcytokine receptor. Features of the Zcytor16 polypeptide include putativesignal sequences at amino acid residues 1 to 21, or 1 to 22 of SEQ IDNO:2, and a mature soluble receptor polyeptpide from residues 22 to 231or 23 to 231 of SEQ ID NO:2. The receptor has two fibronectin IIIdomains, also called immunoglobulin superfamily (Ig) domains,characteristic of the class II cytokine receptor family that compriseamino acid residues 32 to 123 (fibronectin III domain I), and 132 to 230(fibronectin III domain II) of SEQ ID NO:2, and a linker that residesbetween the Ig domains (i.e., at amino acid residues 128-131 of SEQ IDNO:2). Thus molecules of the present invention include polypepetidesthat include a cytokine binding domain comprising amino acids 32 to 230of SEQ ID NO:2. Moreover, additional variants of the zcytor16polypeptide include polyepeptides that comprise amino acid residues 28to 123, 23 to 123, 22 to 123, or 32 to 127, 28 to 127, 23 to 127, 22 to127, (fibronectin III domain I), and 132 to 230 or 231 (fibronectin IIIdomain II) of SEQ ID NO:2, and a linker that resides between the Igdomains (i.e., at amino acid residues 124-131, or 128-131 of SEQ IDNO:2). Thus molecules of the present invention include polypepetidesthat include a cytokine binding domain comprising amino acids 22, 23, or28 to 132 to 230 or 231 of SEQ ID NO:2. In addition, zcytor16 containsconserved motifs and residues characteristic of class II cytokines: anSXWS (SEQ ID NO:46) motif from residue 220-223 of SEQ ID NO:2; conservedTryptophan residues at residues 47, 72, and 114 of SEQ ID NO:2; andconserved Cysteine residues at residues 78, 86, 206, and 227 of SEQ IDNO:2. The Zcytor16 gene is expressed in monocytes, lymphoid, placenta,spleen, tonsil and other tissues, and resides in human chromosome6q23-q24.

The corresponding polynucleotides encoding the zcytor16 polypeptideregions, domains, motifs, residues and sequences described above are asshown in SEQ ID NO:1 (human zcytor16) and SEQ ID NO:37, and SEQ ID NO:47(mouse zcytor16).

As described below, the present invention provides isolated polypeptidescomprising an amino acid sequence that is at least 70%, at least 80%, orat least 90% identical to a reference amino acid sequence of SEQ IDNO:38 or SEQ ID NO:48 selected from the group consisting of: (a) aminoacid residues amino acid residues 24 to 230, or 27 to 230, (b) aminoacid residues 27 to 126, (c) amino acid residues 131 to 230 and (d)amino acid residues amino acid residues 1 to 230. The present inventionalso provides isolated polypeptides as disclosed above that specificallybind with an antibody that specifically binds with a polypeptideconsisting of the amino acid sequence of SEQ ID NO:38 or SEQ ID NO:48.Illustrative polypeptides include polypeptides comprising either aminoacid residues 24 to 230, or 27 to 230 of SEQ ID NO:38 or SEQ ID NO:48 ora fragment thereof described herein. Moreover, the present inventionalso provides isolated polypeptides as disclosed above that bind IL-TIF(e.g., human IL-TIF polypeptide sequence as shown in SEQ ID NO:15 ormouse IL-TIF polypeptide sequence as shown in SEQ ID NO:41). The humanIL-TIF polynucleotide sequence is shown in SEQ ID NO:14. The mouseIL-TIF polynucleotide sequence is shown in SEQ ID NO:40.

The present invention also provides isolated polypeptides comprising atleast 15 contiguous amino acid residues of an amino acid sequence of SEQID NO:38 or SEQ ID NO:48 selected from the group consisting of: (a)amino acid residues amino acid residues 24 to 230, or 27 to 230, (b)amino acid residues 27 to 126, (c) amino acid residues 131 to 230 and(d) amino acid residues amino acid residues 1 to 230. Illustrativepolypeptides include polypeptides that either comprise, or consist of,amino acid residues (a) to (d). Moreover, the present invention alsoprovides isolated polypeptides as disclosed above that bind IL-TIF.

The present invention also includes variant mouse Zcytor16 polypeptides,wherein the amino acid sequence of the variant polypeptide shares anidentity with amino acid residues 24 to 230, or 27 to 230 of SEQ IDNO:38 or SEQ ID NO:48 selected from the group consisting of at least 70%identity, at least 80% identity, at least 90% identity, at least 95%identity, or greater than 95% identity, and wherein any differencebetween the amino acid sequence of the variant polypeptide and thecorresponding amino acid sequence of SEQ ID NO:38 or SEQ ID NO:48 is dueto one or more conservative amino acid substitutions. Moreover, thepresent invention also provides isolated polypeptides as disclosed abovethat bind IL-TIF.

The present invention further provides antibodies and antibody fragmentsthat specifically bind with such polypeptides. Exemplary antibodiesinclude polyclonal antibodies, murine monoclonal antibodies, humanizedantibodies derived from murine monoclonal antibodies, and humanmonoclonal antibodies. Illustrative antibody fragments include F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv, and minimal recognition units. The presentinvention further includes compositions comprising a carrier and apeptide, polypeptide, or antibody described herein.

The present invention also provides isolated nucleic acid molecules thatencode a mouse Zcytor16 polypeptide, wherein the nucleic acid moleculeis selected from the group consisting of: (a) a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:39 or SEQ ID NO:49, (b)a nucleic acid molecule encoding an amino acid sequence that compriseseither amino acid residues 24 to 230, or 27 to 230 of SEQ ID NO:38 orSEQ ID NO:48 and (c) a nucleic acid molecule that remains hybridizedfollowing stringent wash conditions to a nucleic acid moleculecomprising the nucleotide sequence of nucleotides 77 or 86 to 697 of SEQID NO:37 or SEQ ID NO:47, or the complement of the nucleotide sequenceof nucleotides 77 or 86 to 697 of SEQ ID NO:37 or SEQ ID NO:47.Illustrative nucleic acid molecules include those in which anydifference between the amino acid sequence encoded by nucleic acidmolecule (c) and the corresponding amino acid sequence of SEQ ID NO:38or SEQ ID NO:48 is due to a conservative amino acid substitution. Thepresent invention further contemplates isolated nucleic acid moleculesthat comprise nucleotides 8 to 697 of SEQ ID NO:37 or SEQ ID) NO:47.Moreover, the present invention also provides isolated polynucleotidesthat encode polypeptides as disclosed above that bind IL-TIF.

The present invention also includes vectors and expression vectorscomprising such nucleic acid molecules. Such expression vectors maycomprise a transcription promoter, and a transcription terminator,wherein the promoter is operably linked with the nucleic acid molecule,and wherein the nucleic acid molecule is operably linked with thetranscription terminator. The present invention further includesrecombinant host cells and recombinant viruses comprising these vectorsand expression vectors. Illustrative host cells include bacterial,yeast, fungal, insect, mammalian, and plant cells. Recombinant hostcells comprising such expression vectors can be used to produce mouseZcytor16 polypeptides by culturing such recombinant host cells thatcomprise the expression vector and that produce the mouse Zcytor16protein, and, optionally, isolating the mouse Zcytor16 protein from thecultured recombinant host cells.

In addition, the present invention provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and at least one ofsuch an expression vector or recombinant virus comprising suchexpression vectors. The present invention further includespharmaceutical compositions, comprising a pharmaceutically acceptablecarrier and a polypeptide described herein.

The present invention also contemplates methods for detecting thepresence of mouse Zcytor16 RNA in a biological sample, comprising thesteps of (a) contacting a mouse Zcytor16 nucleic acid probe underhybridizing conditions with either (i) test RNA molecules isolated fromthe biological sample, or (ii) nucleic acid molecules synthesized fromthe isolated RNA molecules, wherein the probe has a nucleotide sequencecomprising a portion of the nucleotide sequence of SEQ ID NO:37 or SEQID NO:47, or its complement, and (b) detecting the formation of hybridsof the nucleic acid probe and either the test RNA molecules or thesynthesized nucleic acid molecules, wherein the presence of the hybridsindicates the presence of mouse Zcytor16 RNA in the biological sample.For example, suitable probes consist of the following nucleotidesequences of SEQ ID NO:37 or SEQ ID NO:47: nucleotides 77, 86, or 100 to373; nucleotides 77, 86, or 100 to 694 or 697; nucleotides 401 to 694 or697; and nucleotides 8 to 694 or 697. Other suitable probes consist ofthe complement of these nucleotide sequences, or a portion of thenucleotide sequences or their complements.

The present invention further provides methods for detecting thepresence of mouse Zcytor16 polypeptide in a biological sample,comprising the steps of: (a) contacting the biological sample with anantibody or an antibody fragment that specifically binds with apolypeptide consisting of the amino acid sequence of SEQ ID NO:38 or SEQID NO:48, wherein the contacting is performed under conditions thatallow the binding of the antibody or antibody fragment to the biologicalsample, and (b) detecting any of the bound antibody or bound antibodyfragment. Such an antibody or antibody fragment may further comprise adetectable label selected from the group consisting of radioisotope,fluorescent label, chemiluminescent label, enzyme label, bioluminescentlabel, and colloidal gold.

The present invention also provides kits for performing these detectionmethods. For example, a kit for detection of Zcytor16 gene expressionmay comprise a container that comprises a nucleic acid molecule, whereinthe nucleic acid molecule is selected from the group consisting of (a) anucleic acid molecule comprising the nucleotide sequence of nucleotides77, 86, or 100 to 694 or 697, or 8 to 694 or 697 of SEQ ID NO:37 or SEQID NO:47, (b) a nucleic acid molecule that is a fragment of (a)consisting of at least eight nucleotides. Such a kit may also comprise asecond container that comprises one or more reagents capable ofindicating the presence of the nucleic acid molecule. On the other hand,a kit for detection of mouse Zcytor16 protein may comprise a containerthat comprises an antibody, or an antibody fragment, that specificallybinds with a polypeptide consisting of the amino acid sequence of SEQ IDNO:38 or SEQ ID NO:48.

The present invention also contemplates anti-idiotype antibodies, oranti-idiotype antibody fragments, that specifically bind an antibody orantibody fragment that specifically binds a polypeptide consisting ofthe amino acid sequence of SEQ ID NO:38 or SEQ ID NO:48. An exemplaryanti-idiotype antibody binds with an antibody that specifically binds apolypeptide consisting of amino acid residues 24 to 230, or 27 to 230 ofSEQ ID NO:38 or SEQ ID NO:48.

The present invention also provides isolated nucleic acid moleculescomprising a nucleotide sequence that encodes a mouse Zcytor16 secretionsignal sequence and a nucleotide sequence that encodes a biologicallyactive polypeptide, wherein the mouse Zcytor16 secretion signal sequencecomprises an amino acid sequence of residues 1 to 23 of SEQ ID NO:38 orSEQ ID NO:48. Illustrative biologically active polypeptides includeFactor VIIa, proinsulin, insulin, follicle stimulating hormone, tissuetype plasminogen activator, tumor necrosis factor, interleukin, colonystimulating factor, interferon, erythropoietin, and thrombopoietin.Moreover, the present invention provides fusion proteins comprising amouse Zcytor16 secretion signal sequence and a polypeptide, wherein themouse Zcytor16 secretion signal sequence comprises an amino acidsequence of residues 1 to 23, of SEQ ID NO:38 or SEQ ID NO:48.

The present invention also provides fusion proteins, comprising a mouseZcytor16 polypeptide and an immunoglobulin moiety. In such fusionproteins, the immunoglobulin moiety may be an immunoglobulin heavy chainconstant region, such as a human F_(c) fragment. The present inventionfurther includes isolated nucleic acid molecules that encode such fusionproteins.

The present invention also provides monomeric, homodimeric,heterodimeric and multimeric receptors comprising a mouse Zcytor16extracellular domain. Such receptors are soluble or membrane bound, andact as antagonists of the Zcytor16 ligand, IL-TIF (e.g., the humanIL-TIF as shown in SEQ ID NO:15, and mouse IL-TIF shown in SEQ IDNO:41). In a preferred embodiment, such receptors are soluble receptorscomprising at least one mouse Zcytor16 extracellular domain polypeptidecomprising amino acids 24 to 230, or 27 to 230 of SEQ ID NO:38 or SEQ IDNO:48. The present invention further includes isolated nucleic acidmolecules that encode such receptor polypeptides.

The present invention also provides polyclonal and monoclonal antibodiesto monomeric, homodimeric, heterodimeric and multimeric receptorscomprising a mouse Zcytor16 extracellular domain such as those describedabove. Moreover, such antibodies can be used antagonize the binding tothe Zcytor16 ligand, IL-TIF (SEQ ID NO:15, and SEQ ID NO:41), to theZcytor16 receptor.

The present invention also provides a method for detecting a cancer in apatient, comprising: obtaining a tissue or biological sample from apatient; incubating the tissue or biological sample with an antibody asdescribed above under conditions wherein the antibody binds to itscomplementary polypeptide in the tissue or biological sample;visualizing the antibody bound in the tissue or biological sample; andcomparing levels of antibody bound in the tissue or biological samplefrom the patient to a normal control tissue or biological sample,wherein an increase in the level of antibody bound to the patient tissueor biological sample relative to the normal control tissue or biologicalsample is indicative of a cancer in the patient.

The present invention also provides a method for detecting a cancer in apatient, comprising: obtaining a tissue or biological sample from apatient; labeling a polynucleotide comprising at least 14 contiguousnucleotides of SEQ ID NO:37 or SEQ ID NO:47 or the complement of SEQ IDNO:37 or SEQ ID NO:47; incubating the tissue or biological sample withunder conditions wherein the polynucleotide will hybridize tocomplementary polynucleotide sequence; visualizing the labeledpolynucleotide in the tissue or biological sample; and comparing thelevel of labeled a polynucleotide hybridization in the tissue orbiological sample from the patient to a normal control tissue orbiological sample, wherein an increase in the labeled polynucleotidehybridization to the patient tissue or biological sample relative to thenormal control tissue or biological sample is indicative of a cancer inthe patient.

These and other aspects of the invention will become evident uponreference to the following detailed description. In addition, variousreferences are identified below and are incorporated by reference intheir entirety.

2. Definitions

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,(α-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have alterations in sugarmoieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carbocyclic sugar analogs. Examplesof modifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called, “peptide nucleic acids,” whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

The term “complement of a nucleic acid molecule” refers to a nucleicacid molecule having a complementary nucleotide sequence and reverseorientation as compared to a reference nucleotide sequence. For example,the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

The term “contig” denotes a nucleic acid molecule that has a contiguousstretch of identical or complementary sequence to another nucleic acidmolecule. Contiguous sequences are said to “overlap” a given stretch ofa nucleic acid molecule either in their entirety or along a partialstretch of the nucleic acid molecule.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons as compared to areference nucleic acid molecule that encodes a polypeptide. Degeneratecodons contain different triplets of nucleotides, but encode the sameamino acid residue (i.e., GAU and GAC triplets each encode Asp).

The term “structural gene” refers to a nucleic acid molecule that istranscribed into messenger RNA (mRNA), which is then translated into asequence of amino acids characteristic of a specific polypeptide.

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a growth factor that has been separated from thegenomic DNA of a cell is an isolated DNA molecule. Another example of anisolated nucleic acid molecule is a chemically-synthesized nucleic acidmolecule that is not integrated in the genome of an organism. A nucleicacid molecule that has been isolated from a particular species issmaller than the complete DNA molecule of a chromosome from thatspecies.

A “nucleic acid molecule construct” is a nucleic acid molecule, eithersingle- or double-stranded, that has been modified through humanintervention to contain segments of nucleic acid combined and juxtaposedin an arrangement not existing in nature.

“Linear DNA” denotes non-circular DNA molecules having free 5′ and 3′ends. Linear DNA can be prepared from closed circular DNA molecules,such as plasmids, by enzymatic digestion or physical disruption.

“Complementary DNA (cDNA)” is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand. The term “cDNA” also refers to a clone of a cDNA moleculesynthesized from an RNA template.

“Probes and/or primers” as used herein can be RNA or DNA. DNA can beeither cDNA or genomic DNA. Polynucleotide probes and primers are singleor double-stranded DNA or RNA, generally synthetic oligonucleotides, butmay be generated from cloned cDNA or genomic sequences or itscomplements. Analytical probes will generally be at least 20 nucleotidesin length, although somewhat shorter probes (14-17 nucleotides) can beused. PCR primers are at least 5 nucleotides in length, preferably 15 ormore nt, more preferably 20-30 nt. Short polynucleotides can be usedwhen a small region of the gene is targeted for analysis. For grossanalysis of genes, a polynucleotide probe may comprise an entire exon ormore. Probes can be labeled to provide a detectable signal, such as withan enzyme, biotin, a radionuclide, fluorophore, chemiluminescer,paramagnetic particle and the like, which are commercially availablefrom many sources, such as Molecular Probes, Inc., Eugene, Oreg., andAmersham Corp., Arlington Heights, Ill., using techniques that are wellknown in the art.

A “promoter” is a nucleotide sequence that directs the transcription ofa structural gene. Typically, a promoter is located in the 5′ non-codingregion of a gene, proximal to the transcriptional start site of astructural gene. Sequence elements within promoters that function in theinitiation of transcription are often characterized by consensusnucleotide sequences. These promoter elements include RNA polymerasebinding sites, TATA sequences, CAAT sequences, differentiation-specificelements (DSEs; McGehee et al., Mol. Endocrinol. 7:551 (1993)), cyclicAMP response elements (CREs), serum response elements (SREs; Treisman,Seminars in Cancer Biol. 1:47 (1990)), glucocorticoid response elements(GREs), and binding sites for other transcription factors, such asCRE/ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye etal., J. Biol. Chem. 269:25728 (1994)), SPI, cAMP response elementbinding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and octamerfactors (see, in general, Watson et al., eds., Molecular Biology of theGene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), andLemaigre and Rousseau, Biochem. J. 303:1 (1994)). If a promoter is aninducible promoter, then the rate of transcription increases in responseto an inducing agent. In contrast, the rate of transcription is notregulated by an inducing agent if the promoter is a constitutivepromoter. Repressible promoters are also known.

A “core promoter” contains essential nucleotide sequences for promoterfunction, including the TATA box and start of transcription. By thisdefinition, a core promoter may or may not have detectable activity inthe absence of specific sequences that may enhance the activity orconfer tissue specific activity.

A “regulatory element” is a nucleotide sequence that modulates theactivity of a core promoter. For example, a regulatory element maycontain a nucleotide sequence that binds with cellular factors enablingtranscription exclusively or preferentially in particular cells,tissues, or organelles. These types of regulatory elements are normallyassociated with genes that are expressed in a “cell-specific,”“tissue-specific,” or “organelle-specific” manner.

An “enhancer” is a type of regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

“Heterologous DNA” refers to a DNA molecule, or a population of DNAmolecules, that does not exist naturally within a given host cell. DNAmolecules heterologous to a particular host cell may contain DNA derivedfrom the host cell species (i.e., endogenous DNA) so long as that hostDNA is combined with non-host DNA (i.e., exogenous DNA). For example, aDNA molecule containing a non-host DNA segment encoding a polypeptideoperably linked to a host DNA segment comprising a transcriptionpromoter is considered to be a heterologous DNA molecule. Conversely, aheterologous DNA molecule can comprise an endogenious gene operablylinked with an exogenous promoter. As another illustration, a DNAmolecule comprising a gene derived from a wild-type cell is consideredto be heterologous DNA if that DNA molecule is introduced into a mutantcell that lacks the wild-type gene.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides.”

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

A peptide or polypeptide encoded by a non-host DNA molecule is a“heterologous” peptide or polypeptide.

An “integrated genetic element” is a segment of DNA that has beenincorporated into a chromosome of a host cell after that element isintroduced into the cell through human manipulation. Within the presentinvention, integrated genetic elements are most commonly derived fromlinearized plasmids that are introduced into the cells byelectroporation or other techniques. Integrated genetic elements arepassed from the original host cell to its progeny.

A “cloning vector” is a nucleic acid molecule, such as a plasmid,cosmid, or bacteriophage, that has the capability of replicatingautonomously in a host cell. Cloning vectors typically contain one or asmall number of restriction endonuclease recognition sites that allowinsertion of a nucleic acid molecule in a determinable fashion withoutloss of an essential biological function of the vector, as well asnucleotide sequences encoding a marker gene that is suitable for use inthe identification and selection of cells transformed with the cloningvector. Marker genes typically include genes that provide antibioticresistance, e.g., tetracycline resistance or ampicillin resistance.

An “expression vector” is a nucleic acid molecule encoding a gene thatis expressed in a host cell. Typically, an expression vector comprises atranscription promoter, a gene, and a transcription terminator. Geneexpression is usually placed under the control of a promoter, and such agene is said to be “operably linked to” the promoter. Similarly, aregulatory element and a core promoter are operably linked if theregulatory element modulates the activity of the core promoter.

A “recombinant host” is a cell that contains a heterologous nucleic acidmolecule, such as a cloning vector or expression vector. In the presentcontext, an example of a recombinant host is a cell that produces mouseZcytor16 from an expression vector. In contrast, mouse Zcytor16 can beproduced by a cell that is a “natural source” of mouse Zcytor16, andthat lacks an expression vector.

“Integrative transformants” are recombinant host cells, in whichheterologous DNA has become integrated into the genomic DNA of thecells.

A “fusion protein” is a hybrid protein expressed by a nucleic acidmolecule comprising nucleotide sequences of at least twopolynucleotides, genes, or cDNAs. For example, a fusion protein cancomprise at least part of a mouse Zcytor16 polypeptide fused with apolypeptide that binds an affinity matrix. Such a fusion proteinprovides a means to isolate large quantities of mouse Zcytor16 usingaffinity chromatography.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule termed a “ligand.” This interaction mediates theeffect of the ligand on the cell. Receptors can be membrane bound,cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormonereceptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor,growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor,erythropoietin receptor and IL-6 receptor). Membrane-bound receptors arecharacterized by a multi-domain structure comprising an extracellularligand-binding domain and an intracellular effector domain that istypically involved in signal transduction. In certain membrane-boundreceptors, the extracellular ligand-binding domain and the intracellulareffector domain are located in separate polypeptides that comprise thecomplete functional receptor.

In general, the binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell, which in turnleads to an alteration in the metabolism of the cell. Metabolic eventsthat are often linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids.

A “soluble receptor” is a receptor polypeptide that is not bound to acell membrane. Soluble receptors are most commonly ligand-bindingreceptor polypeptides that lack transmembrane and cytoplasmic domains,and other linkage to the cell membrane such as via glycophosphoinositol(gpi). Soluble receptors can comprise additional amino acid residues,such as affinity tags that provide for purification of the polypeptideor provide sites for attachment of the polypeptide to a substrate, orimmunoglobulin constant region sequences. Many cell-surface receptorshave naturally occurring, soluble counterparts that are produced byproteolysis or translated from alternatively spliced mRNAs. Solublereceptors can be monomeric, homodimeric, heterodimeric, or multimeric,with multimeric receptors generally not comprising more than 9 subunits,preferably not comprising more than 6 subunits, and most preferably notcomprising more than 3 subunits. Receptor polypeptides are said to besubstantially free of transmembrane and intracellular polypeptidesegments when they lack sufficient portions of these segments to providemembrane anchoring or signal transduction, respectively. Solublereceptors of class I and class II cytokine receptors generally comprisethe extracellular cytokine binding domain free of a transmsmbrane domainand intracellular domain. For example, representative soluble receptorsinclude a soluble receptor for CRF2-4 (Genbank Accession No. Z17227) asshown in SEQ ID NO:35; a soluble receptor for IL-10OR (Genbank AccessionNo.s U00672 and NM_(—)001558) as shown in SEQ ID NO:36; and a solublereceptor for zcytor11 (U.S. Pat. No. 5,965,704) as shown in SEQ IDNO:34. It is well within the level of one of skill in the art todelineate what sequences of a known class I or class II cytokinesequence comprise the extracellular cytokine binding domain, free of atransmsmbrane domain and, intracellular domain. Moreover, one of skillin the art using the genetic code can readily determine polynucleotidesthat encode such soluble receptor polyptides.

The term “secretory signal sequence” denotes a DNA sequence that encodesa peptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

An “isolated polypeptide” is a polypeptide that is essentially free fromcontaminating cellular components, such as carbohydrate, lipid, or otherproteinaceous impurities associated with the polypeptide in nature.Typically, a preparation of isolated polypeptide contains thepolypeptide in a highly purified form, i.e., at least about 80% pure, atleast about 90% pure, at least about 95% pure, greater than 95% pure, orgreater than 99% pure. One way to show that a particular proteinpreparation contains an isolated polypeptide is by the appearance of asingle band following sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis of the protein preparation and Coomassie Brilliant Bluestaining of the gel. However, the term “isolated” does not exclude thepresence of the same polypeptide in alternative physical forms, such asdimers or alternatively glycosylated or derivatized forms.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “expression” refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a polypeptide encoded by asplice variant of an mRNA transcribed from a gene.

As used herein, the term “immunomodulator” includes cytokines, stem cellgrowth factors, lymphotoxins, co-stimulatory molecules, hematopoieticfactors, and synthetic analogs of these molecules.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity ofless than 10⁹ M⁻¹.

An “anti-idiotype antibody” is an antibody that binds with the variableregion domain of an immunoglobulin. In the present context, ananti-idiotype antibody binds with the variable region of an anti-mouseZcytor16 antibody, and thus, an anti-idiotype antibody mimics an epitopeof mouse Zcytor16.

An “antibody fragment” is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, and the like. Regardless of structure, an antibodyfragment binds with the same antigen that is recognized by the intactantibody. For example, an anti-mouse Zcytor16 monoclonal antibodyfragment binds with an epitope of mouse Zcytor16.

The term “antibody fragment” also includes a synthetic or a geneticallyengineered polypeptide that binds to a specific antigen, such aspolypeptides consisting of the light chain variable region, “Fv”fragments consisting of the variable regions of the heavy and lightchains, recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“scFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region.

A “chimeric antibody” is a recombinant protein that contains thevariable domains and complementary determining regions derived from arodent antibody, while the remainder of the antibody molecule is derivedfrom a human antibody.

“Humanized antibodies” are recombinant proteins in which murinecomplementarity determining regions of a monoclonal antibody have beentransferred from heavy and light variable chains of the murineimmunoglobulin into a human variable domain.

As used herein, a “therapeutic agent” is a molecule or atom which isconjugated to an antibody moiety to produce a conjugate which is usefulfor therapy. Examples of therapeutic agents include drugs, toxins,immunomodulators, chelators, boron compounds, photoactive agents ordyes, and radioisotopes.

A “detectable label” is a molecule or atom which can be conjugated to anantibody moiety to produce a molecule useful for diagnosis. Examples ofdetectable labels include chelators, photoactive agents, radioisotopes,fluorescent agents, paramagnetic ions, or other marker moieties.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075 (1985);Nilsson et al., Methods Enzymol. 198:3 (1991)), glutathione Stransferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)),substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2:95 (1991). DNA molecules encoding affinity tags areavailable from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.).

A. “naked antibody” is an entire antibody, as opposed to an antibodyfragment, which is not conjugated with a therapeutic agent. Nakedantibodies include both polyclonal and monoclonal antibodies, as well ascertain recombinant antibodies, such as chimeric and humanizedantibodies.

As used herein, the term “antibody component” includes both an entireantibody and an antibody fragment.

An “immunoconjugate” is a conjugate of an antibody component with atherapeutic agent or a detectable label.

As used herein, the term “antibody fusion protein” refers to arecombinant molecule that comprises an antibody component and a mouseZcytor16 polypeptide component. Examples of an antibody fusion proteininclude a protein that comprises a mouse Zcytor16 extracellular domain,and either an Fc domain or an antigen-biding region.

A “target polypeptide” or a “target peptide” is an amino acid sequencethat comprises at least one epitope, and that is expressed on a targetcell, such as a tumor cell, or a cell that carries an infectious agentantigen. T cells recognize peptide epitopes presented by a majorhistocompatibility complex molecule to a target polypeptide or targetpeptide and typically lyse the target cell or recruit other immune cellsto the site of the target cell, thereby killing the target cell.

An “antigenic peptide” is a peptide which will bind a majorhistocompatibility complex molecule to form an MHC-peptide complex whichis recognized by a T cell, thereby inducing a cytotoxic lymphocyteresponse upon presentation to the T cell. Thus, antigenic peptides arecapable of binding to an appropriate major histocompatibility complexmolecule and inducing a cytotoxic T cells response, such as cell lysisor specific cytokine release against the target cell which binds orexpresses the antigen. The antigenic peptide can be bound in the contextof a class I or class II major histocompatibility complex molecule, onan antigen presenting cell or on a target cell.

In eukaryotes, RNA polymerase II catalyzes the transcription of astructural gene to produce mRNA. A nucleic acid molecule can be designedto contain an RNA polymerase II template in which the RNA transcript hasa sequence that is complementary to that of a specific mRNA. The RNAtranscript is termed an “anti-sense RNA” and a nucleic acid moleculethat encodes the anti-sense RNA is termed an, “anti-sense gene.”Anti-sense RNA molecules are capable of binding to mRNA molecules,resulting in an inhibition of mRNA translation.

An “anti-sense oligonucleotide specific for mouse Zcytor16” or a “mouseZcytor16 anti-sense oligonucleotide” is an oligonucleotide having asequence (a) capable of forming a stable triplex with a portion of themouse Zcytor16 gene, or (b) capable of forming a stable duplex with aportion of an mRNA transcript of the mouse Zcytor16 gene.

A “ribozyme” is a nucleic acid molecule that contains a catalyticcenter. The term includes RNA enzymes, self-splicing RNAs, self-cleavingRNAs, and nucleic acid molecules that perform these catalytic functions.A nucleic acid molecule that encodes a ribozyme is termed a “ribozymegene.”

An “external guide sequence” is a nucleic acid molecule that directs theendogenous ribozyme, RNase P, to a particular species of intracellularmRNA, resulting in the cleavage of the mRNA by RNase P. A nucleic acidmolecule that encodes an external guide sequence is termed an “externalguide sequence gene.”

The term “variant mouse Zcytor16 gene” refers to nucleic acid moleculesthat encode a polypeptide having an amino acid sequence that is amodification of SEQ ID NO:38 or SEQ ID NO:48. Such variants includenaturally-occurring polymorphisms of mouse Zcytor16 genes, as well assynthetic genes that contain conservative amino acid substitutions ofthe amino acid sequence of SEQ ID NO:38 or SEQ ID NO:48. Additionalvariant forms of mouse Zcytor16 genes are nucleic acid molecules thatcontain insertions or deletions of the nucleotide sequences describedherein. A variant mouse Zcytor16 gene can be identified, for example, bydetermining whether the gene hybridizes with a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO:37 or SEQ ID NO:47, or itscomplement, under stringent conditions.

Alternatively, variant mouse Zcytor16 genes can be identified bysequence comparison. Two amino acid sequences have “100% amino acidsequence identity” if the amino acid residues of the two amino acidsequences are the same when aligned for maximal correspondence.Similarly, two nucleotide sequences have “100%. nucleotide sequenceidentity” if the nucleotide residues of the two nucleotide sequences arethe same when aligned for maximal correspondence. Sequence comparisonscan be performed using standard software programs such as those includedin the LASERGENE bioinformatics computing suite, which is produced byDNASTAR (Madison, Wis.). Other methods for comparing two nucleotide oramino acid sequences by determining optimal alignment are well-known tothose of skill in the art (see, for example, Peruski and Peruski, TheInternet and the New Biology: Tools for Genomic and Molecular Research(ASM Press, Inc. 1997), Wu et al. (eds.), “Information Superhighway andComputer Databases of Nucleic Acids and Proteins,” in Methods in GeneBiotechnology, pages 123-151 (CRC Press, Inc. 1997), and Bishop (ed.),Guide to Human Genome Computing, 2nd Edition (Academic Press, Inc.1998)). Particular methods for determining sequence identity aredescribed below.

Regardless of the particular method used to identify a variant mouseZcytor16 gene or variant mouse Zcytor16 polypeptide, a variant gene orpolypeptide encoded by a variant gene may be functionally characterizedthe ability to bind specifically to an anti-mouse Zcytor16 antibody. Avariant mouse Zcytor16 gene or variant mouse Zcytor16 polypeptide mayalso be functionally characterized the ability to bind to its ligand,IL-TIF, using a biological or biochemical assay described herein.

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

The present invention includes functional fragments of mouse Zcytor16genes. Within the context of this invention; a “functional fragment” ofa mouse Zcytor16 gene refers to a nucleic acid molecule that encodes aportion of a mouse Zcytor16 polypeptide which is a domain describedherein or at least specifically binds with an anti-mouse Zcytor16antibody.

Due to the imprecision of standard analytical methods, molecular weightsand lengths of polymers are understood to be approximate values. Whensuch a value is expressed as “about” X or “approximately”X, the statedvalue of X will be understood to be accurate to ±10%.

3. Production of Mouse Zcytor16 Polynucleotides or Genes

Nucleic acid molecules encoding a mouse Zcytor16 gene can be obtained byscreening a mouse cDNA or genomic library using polynucleotide probesbased upon SEQ ID NO:37 or SEQ ID NO:47. These techniques are standardand well-established.

As an illustration, a nucleic acid molecule that encodes a mouseZcytor16 gene can be isolated from a cDNA library. In this case, thefirst step would be to prepare the cDNA library by isolating RNA from atissue, such as a tissue wherein mouse Zcytor16 is specificallyexpressed, using methods well-known to those of skill in the art. Ingeneral, RNA isolation techniques must provide a method for breakingcells, a means of inhibiting RNase-directed degradation of RNA, and amethod of separating RNA from DNA, protein, and polysaccharidecontaminants. For example, total RNA can be isolated by freezing tissuein liquid nitrogen, grinding the frozen tissue with a mortar and pestleto lyse the cells, extracting the ground tissue with a solution ofphenol/chloroform to remove proteins, and separating RNA from theremaining impurities by selective precipitation with lithium chloride(see, for example, Ausubel et al. (eds.), Short Protocols in MolecularBiology, 3^(rd) Edition, pages 4-1 to 4-6 (John Wiley & Sons 1995)[“Ausubel (1995)”]; Wu et al., Methods in Gene Biotechnology, pages33-41 (CRC Press, Inc. 1997) [“Wu (1997)”]).

Alternatively, total RNA can be isolated by extracting ground tissuewith guanidinium isothiocyanate, extracting with organic solvents, andseparating RNA from contaminants using differential centrifugation (see,for example, Chirgwin et al. Biochemistry 18:52 (1979); Ausubel (1995)at pages 4-1 to 4-6; Wu (1997) at pages 33-41).

In order to construct a cDNA library, poly(A)⁺ RNA must be isolated froma total RNA preparation. Poly(A)⁺ RNA can be isolated from total RNAusing the standard technique of oligo(dT)-cellulose chromatography (see,for example, Aviv and Leder, Proc. Nat'l Acad. Sci. USA 69:1408 (1972);Ausubel (1995) at pages 4-11 to 4-12).

Double-stranded cDNA molecules are synthesized from poly(A)⁺ RNA usingtechniques well-known to those in the art. (see, for example, Wu (1997)at pages 41-46). Moreover, commercially available kits can be used tosynthesize double-stranded cDNA molecules. For example, such kits areavailable from Life Technologies, Inc. (Gaithersburg, Md.), CLONTECHLaboratories, Inc. (Palo Alto, Calif.), Promega Corporation (Madison,Wis.) and STRATAGENE (La Jolla, Calif.).

Various cloning vectors are appropriate for the construction of a cDNAlibrary. For example, a cDNA library can be prepared in a vector derivedfrom bacteriophage, such as a λgt10 vector. See, for example, Huynh etal., “Constructing and Screening cDNA Libraries in λgt10 and λgt11,” inDNA Cloning: A Practical Approach Vol. I, Glover (ed.), page 49 (IRLPress, 1985); Wu (1997) at pages 47-52.

Alternatively, double-stranded cDNA molecules can be inserted into aplasmid vector, such as a pBLUESCRIPT vector (STRATAGENE; La Jolla,Calif.), a LAMDAGEM-4 (Promega Corp.) or other commercially availablevectors. Suitable cloning vectors also can be obtained from the AmericanType Culture Collection (Manassas, Va.).

To amplify the cloned cDNA molecules, the cDNA library is inserted intoa prokaryotic host, using standard techniques. For example, a cDNAlibrary can be introduced into competent E. coli DH5 or DH10B cells,which can be obtained, for example, from Life Technologies, Inc. orGIBCO BRL (Gaithersburg, Md.).

A human genomic library can be prepared by means well known in the art(see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) atpages 307-327). Genomic DNA can be isolated by lysing tissue with thedetergent Sarkosyl, digesting the lysate with proteinase K, clearinginsoluble debris from the lysate by centrifugation, precipitatingnucleic acid from the lysate using isopropanol, and purifyingresuspended DNA on a cesium chloride density gradient.

DNA fragments that are suitable for the production of a genomic librarycan be obtained by the random shearing of genomic DNA or by the partialdigestion of genomic DNA with restriction endonucleases. Genomic DNAfragments can be inserted into a vector, such as a bacteriophage orcosmid vector, in accordance with conventional techniques, such as theuse of restriction enzyme digestion to provide appropriate termini, theuse of alkaline phosphatase treatment to avoid undesirable joining ofDNA molecules, and ligation with appropriate ligases. Techniques forsuch manipulation are well known in the art (see, for example, Ausubel(1995) at pages 5-1 to 5-6; Wu (1997) at pages 307-327).

Alternatively, human genomic libraries can be obtained from commercialsources such as Research Genetics (Huntsville, Ala.) and the AmericanType Culture Collection (Manassas, Va.).

A library containing cDNA or genomic clones can be screened with one ormore polynucleotide probes based upon SEQ ID NO:37 or SEQ ID NO:47,using standard methods (see, for example, Ausubel (1995) at pages 6-1 to6-11).

Nucleic acid molecules that encode a mouse Zcytor16 gene can also beobtained using the polymerase chain reaction (PCR) with oligonucleotideprimers having nucleotide sequences that are based upon the nucleotidesequences of the mouse Zcytor16 gene, as described herein. Generalmethods for screening libraries with PCR are provided by, for example,Yu et al., “Use of the Polymerase Chain Reaction to Screen PhageLibraries,” in Methods in Molecular Biology, Vol. 15: PCR Protocols:Current Methods and Applications, White (ed.), pages 211-215 (HumanaPress, Inc. 1993). Moreover, techniques for using PCR to isolate relatedgenes are described by, for example, Preston, “Use of DegenerateOligonucleotide Primers and the Polymerase Chain Reaction to Clone GeneFamily Members,” in Methods in Molecular Biology, Vol. 15; PCRProtocols: Current Methods and Applications, White (ed.), pages 317-337(Humana Press, Inc. 1993).

Anti-mouse Zcytor16 antibodies, produced as described below, can also beused to isolate DNA sequences that encode mouse Zcytor16 genes from cDNAlibraries. For example, the antibodies can be used to screen λgt11expression libraries, or the antibodies can be used for immunoscreeningfollowing hybrid selection and translation (see, for example, Ausubel(1995) at pages 6-12 to 6-16; Margolis et al., “Screening λ expressionlibraries with antibody and protein probes,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), pages 1-14(Oxford University Press 1995)).

As an alternative, a mouse Zcytor16 gene can be obtained by synthesizingnucleic acid molecules using mutually priming long oligonucleotides andthe nucleotide sequences described herein (see, for example, Ausubel(1995) at pages 8-8 to 8-9). Established techniques using the polymerasechain reaction provide the ability to synthesize DNA molecules at leasttwo kilobases in length (Adang et al., Plant Molec. Biol. 21:1131(1993), Bambot et al., PCR Methods and Applications 2:266 (1993), Dillonet al., “Use of the Polymerase Chain Reaction for the Rapid Constructionof Synthetic Genes,” in Methods in Molecular Biology, Vol. 15: PCRProtocols: Current Methods and Applications, White (ed.), pages 263-268,(Humana Press, Inc. 1993), and Holowachuk et al., PCR Methods Appl.4:299 (1995)).

The nucleic acid molecules of the present invention can also besynthesized with “gene machines” using protocols such as thephosphoramidite method. If chemically-synthesized double stranded DNA isrequired for an application such as the synthesis of a gene or a genefragment, then each complementary strand is made separately. Theproduction of short genes (60 to 80 base pairs) is technicallystraightforward and can be accomplished by synthesizing thecomplementary strands and then annealing them. For the production oflonger genes (>300 base pairs), however, special strategies may berequired, because the coupling efficiency of each cycle during chemicalDNA synthesis is seldom 100%. To overcome this problem, synthetic genes(double-stranded) are assembled in modular form from single-strandedfragments that are from 20 to 100 nucleotides in length. For reviews onpolynucleotide synthesis, see, for, example, Glick and Pasternak,Molecular Biotechnology, Principles and Applications of Recombinant DNA(ASM Press 1994), Itakura et al., Annu. Rev. Biochem. 53:323 (1984), andClimie et al., Proc. Nat'l Acad. Sci. USA 87:633 (1990).

The sequence of a mouse Zcytor16 cDNA or mouse Zcytor16 genomic fragmentcan be determined using standard methods. Mouse Zcytor16 polynucleotidesequences disclosed herein can also be used as probes or primers toclone 5′ non-coding regions of a mouse Zcytor16 gene. Because mouseZcytor16 is expressed in a limited number of specific tissues, promoterelements from a mouse Zcytor16 gene can be used to direct the expressionof heterologous genes in, for example, specific tissues of transgenicanimals or patients treated with gene therapy. The identification ofgenomic fragments containing a mouse Zcytor16 promoter or regulatoryelement can be achieved using well-established techniques, such asdeletion analysis (see, generally, Ausubel (1995)).

Cloning of 5′ flanking sequences also facilitates production of mouseZcytor16 proteins by “gene activation,” as disclosed in U.S. Pat. No.5,641,670. Briefly, expression of an endogenous mouse Zcytor16 gene in acell is altered by introducing into the mouse Zcytor16 locus a DNAconstruct comprising at least a targeting sequence, a regulatorysequence, an exon, and an unpaired splice donor site. The targetingsequence is a mouse Zcytor16 5′ non-coding sequence that permitshomologous recombination of the construct with the endogenous mouseZcytor16 locus, whereby the sequences within the construct becomeoperably linked with the endogenous mouse Zcytor16 coding sequence. Inthis way, an endogenous mouse Zcytor16 promoter can be replaced orsupplemented with other regulatory sequences to provide enhanced,tissue-specific, or otherwise regulated expression.

4. Production of Mouse Zcytor16 Gene Variants

The present invention provides a variety of nucleic acid molecules,including DNA and RNA molecules, that encode the mouse Zcytor16polypeptides disclosed herein. Those skilled in the art will readilyrecognize that, in view of the degeneracy of the genetic code,considerable sequence variation is possible among these polynucleotidemolecules. SEQ ID NO:3 is a degenerate nucleotide sequence thatencompasses all nucleic acid molecules that encode the human Zcytor16polypeptide of SEQ ID NO:2. Similarly, SEQ ID NO:39 is a degeneratenucleotide sequence that encompasses all nucleic acid molecules thatencode the mouse Zcytor16 polypeptide of SEQ ID NO:38; and SEQ ID NO:49is a degenerate nucleotide sequence that encompasses all nucleic acidmolecules that encode the mouse Zcytor16 polypeptide of SEQ ID NO:48.Those skilled in the art will recognize that the degenerate sequence ofSEQ ID NO:39 and SEQ ID NO:49 also provides all RNA sequences encodingSEQ ID NO:38 and SEQ ID NO:48 respectively, by substituting U for T.Moreover, the present invention also provides isolated solublemonomeric, homodimeric, heterodimeric and multimeric receptorpolypeptides that comprise at least one mouse Zcytor16 receptor subunitthat is substantially homologous to the receptor polypeptide of SEQ IDNO:38 or SEQ ID NO:48. Thus, the present invention contemplates mouseZcytor16 polypeptide-encoding nucleic acid molecules comprisingnucleotide 1 to nucleotide 693 of SEQ ID NO:39, and nucleic acidmolecules comprising nucleotide 1 to nucleotide 693 of SEQ ID NO:49, andtheir RNA equivalents. Moreover, mouse zcytor16 fragments describedherein in SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:47 and SEQ ID NO:48 withreference to SEQ ID NO:39 and SEQ ID NO:49, are also contemplated.

Table 1 sets forth the one-letter codes used within SEQ ID NO:39 or SEQID NO:49 to denote degenerate nucleotide positions. “Resolutions” arethe nucleotides denoted by a code letter. “Complement” indicates thecode for the complementary nucleotide(s). For example, the code Ydenotes either C or T, and its complement R denotes A or G, A beingcomplementary to T, and G being complementary to C. TABLE 1 NucleotideResolution Complement Resolution A A T T C C G G G G C C T T A A R A|G YC|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|G W A|T W A|T H A|C|TD A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T H A|C|T N A|C|G|T NA|C|G|T

The degenerate codons used in SEQ ID NO:39 or SEQ ID NO:49, encompassingall possible codons for a given amino acid, are set forth in Table 2.TABLE 2 One Amino Letter Degenerate Acid Code Codons Codon Cys C TGC TGTTGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro PCCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGNAsn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CARHis H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AARMet M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTNVal V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGGTGG Ter · TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding an amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequences of SEQ ID NO:38 or SEQ ID NO:48. Variant sequences can bereadily tested for functionality as described herein.

Different species can exhibit “preferential codon usage.” In general,see, Grantham et al., Nucl. Acids Res. 8:1893 (1980), Haas et al. Curr.Biol. 6:315 (1996), Wain-Hobson et al., Gene 13:355 (1981), Grosjean andFiers, Gene 18:199 (1982), Holm, Nuc. Acids Res. 14:3075 (1986),Ikemura, J. Mol. Biol. 158:573 (1982), Sharp and Matassi, Curr. Opin.Genet. Dev. 4:851 (1994), Kane, Curr. Opin. Biotechnol. 6:494 (1995),and Makrides, Microbiol. Rev. 60:512 (1996). As used herein, the term“preferential codon usage” or “preferential codons” is a term of artreferring to protein translation codons that are most frequently used incells of a certain species, thus favoring one or a few representativesof the possible codons encoding each amino acid (See Table 2). Forexample, the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG,or ACT, but in mammalian cells ACC is the most commonly used codon; inother species, for example, insect cells, yeast, viruses or bacteria,different Thr codons may be preferential. Preferential codons for aparticular species can be introduced into the polynucleotides of thepresent invention by a variety of methods known in the art. Introductionof preferential codon sequences into recombinant DNA can, for example,enhance production of the protein by making protein translation moreefficient within a particular cell type or species. Therefore, thedegenerate codon sequences disclosed herein serve as a template foroptimizing expression of polynucleotides in various cell types andspecies commonly used in the art and disclosed herein. Sequencescontaining preferential codons can be tested and optimized forexpression in various species, and tested for functionality as disclosedherein.

The present invention further provides variant polypeptides and nucleicacid molecules that represent counterparts from other species(orthologs). These species include, but are not limited to mammalian,avian, amphibian, reptile, fish, insect and other vertebrate andinvertebrate species. Of particular interest are Zcytor16 polypeptidesfrom other mammalian species, including human, rat, porcine, ovine,bovine, canine, feline, equine, and other primate polypeptides.Orthologs of mouse Zcytor16 can be cloned using information andcompositions provided by the present invention in combination withconventional cloning techniques. For example, a mouse Zcytor16 cDNA canbe cloned using mRNA obtained from a tissue or cell type that expressesmouse Zcytor16 as disclosed herein. Suitable sources of mRNA can beidentified by probing northern blots with probes designed from thesequences disclosed herein. A library is then prepared from mRNA of apositive tissue or cell line.

A Zcytor16-encoding cDNA can be isolated by a variety of methods, suchas by probing with a complete or partial cDNA or with one or more setsof degenerate probes based on the disclosed sequences. A cDNA can alsobe cloned using the polymerase chain reaction with primers designed fromthe representative mouse Zcytor16 sequences disclosed herein. Inaddition, a cDNA library can he used to transform or transfect hostcells, and expression of the cDNA of interest can be detected with anantibody to mouse Zcytor16 polypeptide.

Those skilled in the art will recognize that the sequence disclosed inSEQ ID NO:37 or SEQ ID NO:47 represents a single allele of mouseZcytor16, and that allelic variation and alternative splicing areexpected to occur. Allelic variants of this sequence can be cloned byprobing cDNA or genomic libraries from different individuals accordingto standard procedures. Allelic variants of the nucleotide sequencesdisclosed herein, including those containing silent mutations and thosein which mutations result in amino acid sequence changes, are within thescope of the present invention, as are proteins which are allelicvariants of the amino acid sequences disclosed herein. cDNA moleculesgenerated from alternatively spliced mRNAs, which retain the propertiesof the mouse Zcytor16 polypeptide are included within the scope of thepresent invention, as are polypeptides encoded by such cDNAs and mRNAs.Allelic variants and splice variants of these sequences can be cloned byprobing cDNA or genomic libraries from different individuals or tissuesaccording to standard procedures known in the art.

Using the methods discussed above, one of ordinary skill in the art canprepare a variety of polypeptides that comprise a soluble receptorsubunit that is substantially homologous to SEQ ID NO:37 or SEQ IDNO:47, or SEQ ID NO:38 or SEQ ID NO:48, amino acids 24 to 230, or 27 to230 of SEQ ID NO:38 or SEQ ID NO:48, or allelic variants thereof andretain the ligand-binding properties of the wild-type mouse Zcytor16receptor. Such polypeptides may also include additional polypeptidesegments as generally disclosed herein.

Within certain embodiments of the invention, the isolated nucleic acidmolecules can hybridize under stringent conditions to nucleic acidmolecules comprising nucleotide sequences disclosed herein. For example,such nucleic acid molecules can hybridize under stringent conditions tonucleic acid molecules comprising the nucleotide sequence of SEQ IDNO:37 or SEQ ID NO:47, to nucleic acid molecules consisting of thenucleotide sequence of nucleotides 8 to 694 or 697; or 77, 86, or 100 to694 or 697 of SEQ ID NO:37 or SEQ ID NO:47, or to nucleic acid moleculescomprising a nucleotide sequence complementary to SEQ ID NO:37 or SEQ IDNO:47 or to nucleotides 8 to. 694 or 697; or 77, 86, or 100 to 694 or697 of SEQ ID NO:37 or SEQ ID NO:47, or fragments thereof. In general,stringent conditions are selected to be about 5° C. lower than thethermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe.

A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA,can hybridize if the nucleotide sequences have some degree ofcomplementarity. Hybrids can tolerate mismatched base pairs in thedouble helix, but the stability of the hybrid is influenced by thedegree of mismatch. The T_(m) of the mismatched hybrid decreases by 1°C. for every 1-1.5% base pair mismatch. Varying the stringency of thehybridization conditions allows control over the degree of mismatch thatwill be present in the hybrid. The degree of stringency increases as thehybridization temperature increases and the ionic strength of thehybridization buffer decreases. Stringent hybridization conditionsencompass temperatures of about 5-25° C. below the T_(m) of the hybridand a hybridization buffer having up to 1 M Na⁺. Higher degrees ofstringency at lower temperatures can be achieved with the addition offormamide which reduces the T_(m) of the hybrid about 1° C. for each 1%formamide in the buffer solution. Generally, such stringent conditionsinclude temperatures of 20-70° C. and a hybridization buffer containingup to 6×SSC and 0-50% formamide. A higher degree of stringency can beachieved at temperatures of from 40-70° C. with a hybridization bufferhaving up to 4×SSC and from 0-50% formamide. Highly stringent conditionstypically encompass temperatures of 42-70° C. with a hybridizationbuffer having up to 1×SSC and 0-50% formamide. Different degrees ofstringency can be used during hybridization and washing to achievemaximum specific binding to the target sequence. Typically, the washesfollowing hybridization are performed at increasing degrees ofstringency to remove non-hybridized polynucleotide probes fromhybridized complexes.

The above conditions are meant to serve as a guide and it is well withinthe abilities of one skilled in the art to adapt these conditions foruse with a particular polynucleotide hybrid. The T_(m) for a specifictarget sequence is the temperature (under defined conditions) at which50% of the target sequence will hybridize to a perfectly matched probesequence. Those conditions which influence the T_(m) include, the sizeand base pair content of the polynucleotide probe, the ionic strength ofthe hybridization solution, and the presence of destabilizing agents inthe hybridization solution. Numerous equations for calculating T_(m) areknown in the art, and are specific for DNA, RNA and DNA-RNA hybrids andpolynucleotide probe sequences of varying length (see, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition(Cold Spring Harbor Press 1989); Ausubel et al., (eds.), CurrentProtocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Bergerand Kimmel (eds.), Guide to Molecular Cloning Techniques, (AcademicPress, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227(1990)). Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake,Minn.) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto,Calif.), as well as sites on the Internet, are available tools foranalyzing a given sequence and calculating T_(m) based on user definedcriteria. Such programs can also analyze a given sequence under definedconditions and identify suitable probe sequences. Typically,hybridization of longer polynucleotide sequences, >50 base pairs, isperformed at temperatures of about 20-25° C. below the calculated T_(m).For smaller probes, <50 base pairs, hybridization is typically carriedout at the T_(m) or 5-10° C. below. This allows for the maximum rate ofhybridization for DNA-DNA and DNA-RNA hybrids.

The length of the polynucleotide sequence influences the rate andstability of hybrid formation. Smaller probe sequences, <50 base pairs,reach equilibrium with complementary sequences rapidly, but may formless stable hybrids. Incubation times of anywhere from minutes to hourscan be used to achieve hybrid formation. Longer probe sequences come toequilibrium more slowly, but form more stable complexes even at lowertemperatures. Incubations are allowed to proceed overnight or longer.Generally, incubations are carried out for a period equal to three timesthe calculated Cot time. Cot time, the time it takes for thepolynucleotide sequences to reassociate, can be calculated for aparticular sequence by methods known in the art.

The base pair composition of polynucleotide sequence will effect thethermal stability of the hybrid complex, thereby influencing the choiceof hybridization temperature and the ionic strength of the hybridizationbuffer. A-T pairs are less stable than G-C pairs in aqueous solutionscontaining sodium chloride. Therefore, the higher the G-C content, themore stable the hybrid. Even distribution of G and C residues within thesequence also contribute positively to hybrid stability. In addition,the base pair composition can be manipulated to alter the T_(m) of agiven sequence. For example, 5-methyldeoxycytidine can be substitutedfor deoxycytidine and 5-bromodeoxuridine can be substituted forthymidine to increase the T_(m), whereas 7-deazz-2′-deoxyguanosine canbe substituted for guanosine to reduce dependence on T_(m).

The ionic concentration of the hybridization buffer also affects thestability of the hybrid. Hybridization buffers generally containblocking agents such as Denhardt's solution (Sigma Chemical Co., St.Louis, Mo.), denatured salmon sperm DNA, tRNA, milk powders (BLOTTO),heparin or SDS, and a Na⁺ source, such as SSC (1×SSC: 0.15 M sodiumchloride, 15 mM sodium citrate) or SSPE (1×SSPE: 1.8 M NaCl, 10 mMNaH₂PO₄, 1 mM EDTA, pH 7.7). Typically, hybridization buffers containfrom between 10 mM-1 M Na⁺. The addition of destabilizing or denaturingagents such as formamide, tetralkylammonium salts, guanidinium cationsor thiocyanate cations to the hybridization solution will alter theT_(m) of a hybrid. Typically, formamide is used at a concentration of upto 50% to allow incubations to be carried out at more convenient andlower temperatures. Formamide also acts to reduce non-specificbackground when using RNA probes.

As an illustration, a nucleic acid molecule encoding a variant mouseZcytor16 polypeptide can be hybridized with a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO:37 or SEQ ID NO:47 (or itscomplement) at 42° C. overnight in a solution comprising 50% formamide,5×SSC, 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution (100×Denhardt's solution: 2% (w/v) Ficoll 400, 2% (w/v) polyvinylpyrrolidone,and 2% (w/v) bovine serum albumin), 10% dextran sulfate, and 20 μg/mldenatured, sheared salmon sperm DNA. One of skill in the art can devisevariations of these hybridization conditions. For example, thehybridization mixture can be incubated at a higher temperature, such asabout 65° C., in a solution that does,not contain formamide. Moreover,premixed hybridization solutions are available (e.g., EXPRESSHYBHybridization Solution from CLONTECH Laboratories, Inc.), andhybridization can be performed according to the manufacturer'sinstructions.

Following hybridization, the nucleic acid molecules can be washed toremove non-hybridized nucleic acid molecules under stringent conditions,or under highly stringent conditions. Typical stringent washingconditions include washing in a solution of 0.5×-2×SSC with 0.1% sodiumdodecyl sulfate (SDS) at 55-65° C. As an illustration, nucleic acidmolecules encoding a variant mouse Zcytor16 polypeptide remainhybridized with a nucleic acid molecule having the nucleotide sequenceof SEQ ID NO:37 or SEQ ID NO:47 (or its complement) under stringentwashing conditions, in which the wash stringency is equivalent to0.5×-2×SSC with 0.1% SDS at 55-65° C., including 0.5×SSC with 0.1% SDSat 55° C., or 2×SSC with 0.1% SDS at 65° C. One of skill in the art canreadily devise equivalent conditions, for example, by substituting SSPEfor SSC in the wash solution.

Typical highly stringent washing conditions include washing in asolution of 0.1×-0.2×SSC with 0.1% sodium dodecyl sulfate (SDS) at50-65° C. For example, nucleic acid molecules encoding a variant mouseZcytor16 polypeptide remain hybridized with a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO:37 or SEQ ID NO:47 (or itscomplement) under highly stringent washing conditions, in which the washstringency is equivalent to 0.1×-0.2×SSC with 0.1% SDS at 50-65° C.,including 0.1×SSC with 0.1% SDS at 50° C., or 0.2×SSC with 0.1% SDS at65° C.

The present invention also provides isolated mouse Zcytor16 polypeptidesthat have a substantially similar sequence identity to the polypeptidesof SEQ ID NO:38 or SEQ ID NO:48, or their orthologs. The term“substantially similar sequence identity” is used herein to denotepolypeptides having at least 70%, at least 80%, at least 90%, at least95% or greater than 95% sequence identity to the sequences shown in SEQID NO:38 or SEQ ID NO:48, or their orthologs.

The present invention also contemplates mouse Zcytor16 variant nucleicacid molecules that can be identified using two criteria: adetermination of the similarity between the encoded polypeptide with theamino acid sequence of SEQ ID NO:38 or SEQ ID NO:48, and a hybridizationassay, as described above. Such mouse Zcytor16 variants include nucleicacid molecules (1) that remain hybridized with a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO:37 or SEQ ID NO:47 (or itscomplement) under stringent washing conditions, in which the washstringency is equivalent to 0.5×-2×SSC with 0.1% SDS at 55-65° C., and(2) that encode a polypeptide having at least 70%, at least 80%, atleast 90%, at least 95% or greater than 95% sequence identity to theamino acid sequence of SEQ ID NO:38 or SEQ ID NO:48. Alternatively,mouse Zcytor16 variants can be characterized as nucleic acid molecules(1) that remain hybridized with a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:37 or SEQ ID NO:47 (or its complement)under highly stringent washing conditions, in which the wash stringencyis equivalent to 0.1×-0.2×SSC with 0.1% SDS at 50-65° C., and (2) thatencode a polypeptide having at least 70%, at least 80%, at least 90%, atleast 95% or greater than 95% sequence identity to the amino acidsequence of SEQ ID NO:38 or SEQ ID NO:48.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 3 (amino acids are indicated by the standard one-lettercodes). The percent identity is then calculated as: ([Total number ofidentical matches]/[length of the longer sequence plus the number ofgaps introduced into the longer sequence in order to align the twosequences])(100). TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R−1 5 N −2 0 6 D −2 −2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 25 G 0 −2 0 −1 −3 −2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3−4 −3 4 L −1 −2 −3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −25 M −1 −1 −2 −3 −1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0−3 0 6 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0−1 −2 −2 0 −1 −2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W−3 −3 −4 −4 −2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1−2 −3 2 −1 −1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1−1 −2 −2 0 −3 −1 4

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativemouse Zcytor16 variant. The FASTA algorithm is described by Pearson andLipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990). Briefly, FASTA first characterizes sequencesimilarity by identifying regions shared by the query sequence (e.g.,SEQ ID NO:38 or SEQ ID NO:48) and a test sequence that have either thehighest density of identities (if the ktup variable is 1) or pairs ofidentities (if ktup=2), without considering conservative amino acidsubstitutions, insertions, or deletions. The ten regions with thehighest density of identities are then rescored by comparing thesimilarity of all paired amino acids using an amino acid substitutionmatrix, and the ends of the regions are “trimmed” to include only thoseresidues that contribute to the highest score. If there are severalregions with scores greater than the “cutoff” value (calculated by apredetermined formula based upon the length of the sequence and the ktupvalue), then the trimmed initial regions are examined to determinewhether the regions can be joined to form an approximate alignment withgaps. Finally, the highest scoring regions of the two amino acidsequences are aligned using a modification of theNeedleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allowsfor amino acid insertions and deletions. Illustrative parameters forFASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other parameters set asdescribed above.

The present invention includes nucleic acid molecules that encode apolypeptide having a conservative amino acid change, compared with anamino acid sequence disclosed herein. For example, variants can beobtained that contain one or more amino acid substitutions of SEQ IDNO:38 or SEQ ID NO:48, in which an alkyl amino acid is substituted foran alkyl amino acid in a mouse Zcytor16 amino acid sequence, an aromaticamino acid is substituted for an aromatic amino acid in a mouse Zcytor16amino acid sequence, a sulfur-containing amino acid is substituted for asulfur-containing amino acid in a mouse Zcytor16 amino acid sequence, ahydroxy-containing amino acid is substituted for a hydroxy-containingamino acid in a mouse Zcytor16 amino acid sequence, an acidic amino acidis substituted for an acidic amino acid in a mouse Zcytor16 amino acidsequence, a basic amino acid is substituted for a basic amino acid in amouse Zcytor16 amino acid sequence, or a dibasic monocarboxylic aminoacid is substituted for a dibasic monocarboxylic amino acid in a mouseZcytor16 amino acid sequence. Among the common amino acids, for example,a “conservative amino acid substitution” is illustrated by asubstitution among amino acids within each of the following groups: (1)glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine,tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate andglutamate, (5) glutamine and asparagine, and (6) lysine, arginine andhistidine The BLOSUM62 table is an amino acid substitution matrixderived from about 2,000 local multiple alignments of protein sequencesegments, representing highly conserved regions of more than 500 groupsof related proteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA89:10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies canbe used to define conservative amino acid substitutions that may beintroduced into the amino acid sequences of the present invention.Although it is possible to design amino acid substitutions based solelyupon chemical properties (as discussed above), the language“conservative amino acid substitution” preferably refers to asubstitution represented by a BLOSUM62 value of greater than −1. Forexample, an amino acid substitution is conservative if the substitutionis characterized by a BLOSUM62 value of 0, 1, 2, or 3. According to thissystem, preferred conservative amino acid substitutions arecharacterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), whilemore preferred conservative amino acid substitutions are characterizedby a BLOSUM62 value of at least 2 (e.g., 2 or 3).

Particular variants of mouse Zcytor16 are characterized by having atleast 70%, at least 80%, at least 90%, at least 95% or greater than 95%sequence identity to the corresponding amino acid sequence (e.g., SEQ IDNO:38 or SEQ ID NO:48), wherein the variation in amino acid sequence isdue to one or more conservative amino acid substitutions.

Conservative amino acid changes in a mouse Zcytor16 gene can beintroduced, for example, by substituting nucleotides for the nucleotidesrecited in SEQ ID NO:37 or SEQ ID NO:47. Such “conservative amino acid”variants can be obtained by oligonucleotide-directed mutagenesis,linker-scanning mutagenesis, mutagenesis using the polymerase chainreaction, and the like (see Ausubel (1995) at pages 8-10 to 8-22; andMcPherson (ed.), Directed Mutagenesis: A Practical Approach (IRL Press1991)). A variant mouse Zcytor16 polypeptide can be identified by theability to specifically bind anti-mouse Zcytor16 antibodies.

The proteins of the present invention can also comprise non-naturallyoccurring amino acid residues. Non-naturally occurring amino acidsinclude, without limitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Transcriptionand translation of plasmids containing nonsense mutations is typicallycarried out in a cell-free system comprising an E. coli S30 extract andcommercially available enzymes and other reagents. Proteins are purifiedby chromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722 (1991), Ellman et al., Methods Enzymol. 202:301 (1991), Chunget al., Science 259:806 (1993), and Chung et al., Proc. Nat'l Acad. Sci.USA 90:10145 (1993).

In a second method, translation is carried out in Xenopus oocytes bymicroinjection of mutated mRNA and chemically aminoacylated suppressortRNAs (Turcatti et al., J. Biol. Chem. 271:19991 (1996)). Within a thirdmethod, E. coli cells are cultured in the absence of a natural aminoacid that is to be replaced (e.g., phenylalanine) and in the presence ofthe desired non-naturally occurring amino acid(s) (e.g.,2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or4-fluorophenylalanine). The non-naturally occurring amino acid isincorporated into the protein in place of its natural counterpart. See,Koide et al., Biochem. 33:7470 (1994). Naturally occurring amino acidresidues can be converted to non-naturally occurring species by in vitrochemical modification. Chemical modification can be combined withsite-directed mutagenesis to further expand the range of substitutions(Wynn and Richards, Protein Sci. 2:395 (1993)).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for mouse Zcytor16 aminoacid residues.

Essential amino acids in the polypeptides of the present invention canbe identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081 (1989), Bass et al., Proc. Nat'l Acad. Sci.USA 88:4498 (1991), Coombs and Corey, “Site-Directed Mutagenesis andProtein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.),pages 259-311 (Academic Press, Inc. 1998)). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalactivity to identify amino acid residues that are critical to theactivity of the molecule. See also, Hilton et al., J. Biol. Chem.271:4699 (1996).

Although sequence analysis can be used to further define the Zcytor16ligand binding region, amino acids that play a role in mouse Zcytor16binding activity (such as binding of mouse Zcytor16 to ligand IL-TIF, orto an anti-mouse Zcytor16 antibody) can also be determined by physicalanalysis of structure, as determined by such techniques as nuclearmagnetic resonance, crystallography, electron diffraction orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., Science 255:306(1992), Smith et al., J. Mol. Biol. 224:899 (1992), and Wlodaver et al.,FEBS Lett. 309:59 (1992).

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53 (1988)) or Bowie and Sauer(Proc. Nat'l Acad. Sci. USA 86:2152 (1989)). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner etal., U.S. Pat. No. 5,223,409, Huse, international publication No. WO92/06204, and region-directed mutagenesis (Derbyshire et al., Gene46:145 (1986), and Ner et al., DNA 7:127, (1988)). Moreover, mouseZcytor16 labeled with biotin or FITC can be used for expression cloningof Zcytor16 ligands.

Variants of the disclosed mouse Zcytor16 nucleotide and polypeptidesequences can also be generated through DNA shuffling as disclosed byStemmer, Nature 370:389 (1994), Stemmer, Proc. Nat'l Acad. Sci. USA91:10747 (1994), and international publication No. WO 97/20078. Briefly,variant DNA molecules are generated by in vitro homologous recombinationby random fragmentation of a parent DNA followed by reassembly usingPCR, resulting in randomly introduced point mutations. This techniquecan be modified by using a family of parent DNA molecules, such asallelic variants or DNA molecules from different species, to introduceadditional variability into the process. Selection or screening for thedesired activity, followed by additional iterations of mutagenesis andassay provides for rapid “evolution” of sequences by selecting fordesirable mutations while simultaneously selecting against detrimentalchanges.

Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode biologically active polypeptides, or polypeptidesthat bind with anti-mouse Zcytor16 antibodies, can be recovered from thehost cells and rapidly sequenced using modern equipment. These methodsallow the rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

The present invention also includes “functional fragments” of mouseZcytor16 polypeptides and nucleic acid molecules encoding suchfunctional fragments. Routine deletion analyses of nucleic acidmolecules can be performed to obtain functional fragments of a nucleicacid molecule that encodes a mouse Zcytor16 polypeptide. As anillustration, DNA molecules having the nucleotide sequence of SEQ IDNO:37 or SEQ ID NO:47 can be digested with Bal31 nuclease to obtain aseries of nested deletions. The fragments are then inserted intoexpression vectors in proper reading frame, and the expressedpolypeptides are isolated and tested for the ability to bind anti-mouseZcytor16 antibodies. One alternative to exonuclease digestion is to useoligonucleotide-directed mutagenesis to introduce deletions or stopcodons to specify production of a desired fragment. Alternatively,particular fragments of a mouse Zcytor16 gene can be synthesized usingthe polymerase chain reaction.

This general approach is exemplified by studies on the truncation ateither or both termini of interferons have been summarized byHorisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover,standard techniques for functional analysis of proteins are describedby, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993),Content et al., “Expression and preliminary deletion analysis of the 42kDa 2-SA synthetase induced by human interferon,” in BiologicalInterferon Systems, Proceedings of ISIR-TNO Meeting on InterferonSystems, Cantell (ed.), pages 65-72 (Nijhoff 1987), Herschman, “The EGFReceptor,” in Control of Animal Cell Proliferation, Vol. 1, Boynton etal., (eds.) pages 169-199 (Academic Press 1985), Coumailleau et al., J.Biol. Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291(1995); Yamaguchi et al., Biochem. Pharmacol. 50:1295 (1995), and Meiselet al., Plant Molec. Biol. 30:1 (1996).

Analysis of the particular sequences disclosed herein provide a set ofillustrative functional fragments presented in Table 4, wherein “F_(III)Domain” is used to denote fibronectin III domains. The nucleotidesencoding additional mouse zcytor16 functional varaint domains describedherein, not show in Table 4, can be determined with reference to SEQ IDNO:37 or SEQ ID NO:47. Such functional fragments include for example,the following nucleotide sequences of SEQ ID NO:37 or SEQ ID NO:47:nucleotides 77, 86, or 100 to 373; nucleotides 77, 86, or 100 to 694 or697; nucleotides 401 to 694 or 697; and nucleotides 8 to 694 or 697, andamino acid sequences encoded thereby, e.g, such as those shown in SEQ IDNO:38 or SEQ ID NO:48 respectively. TABLE 4 Amino acid residuesNucleotides Mouse Zcytor16 (SEQ ID NO: 38 or (SEQ ID NO: 37 or FeatureSEQ ID NO: 48) SEQ ID NO: 47) First F_(III) Domain 31-122 100-373 SecondF_(III) Domain 131-229  401-694 Both F_(III) Domains 31-229 100-694

The present invention also contemplates functional fragments of a mouseZcytor16 gene that have amino acid changes, compared with an amino acidsequence disclosed herein. A variant mouse Zcytor16 gene can beidentified on the basis of structure by determining the level ofidentity with disclosed nucleotide and amino acid sequences, asdiscussed above. An alternative approach to identifying a variant geneon the basis of structure is to determine whether a nucleic acidmolecule encoding a potential variant mouse Zcytor16 gene can hybridizeto a nucleic acid molecule comprising a nucleotide sequence, such as SEQID NO:37 or SEQ ID NO:47.

For any mouse Zcytor16 polypeptide, including variants and fusionproteins, one of ordinary skill in the art can readily generate a fullydegenerate polynucleotide sequence encoding that variant using theinformation set forth in Tables 1 and 2 above. Moreover, those of skillin the art can use standard software to devise mouse Zcytor16 variantsbased upon the nucleotide and amino acid sequences described herein.Accordingly, the present invention includes a computer-readable mediumencoded with a data structure that provides at least one of thefollowing sequences: SEQ ID NO:37 or SEQ ID NO:47, SEQ ID NO:38 or SEQID NO:48, and SEQ ID NO:39 or SEQ ID NO:49. Suitable forms ofcomputer-readable media include magnetic media and optically-readablemedia. Examples of magnetic media include a hard or fixed drive, arandom access memory (RAM) chip, a floppy disk, digital linear tape(DLT), a disk cache, and a ZIP disk. Optically readable media areexemplified by compact discs (e.g., CD-read only memory (ROM),CD-rewritable (RW), and CD-recordable), and digital versatile/videodiscs (DVD) (e.g., DVD−ROM, DVD−RAM, and DVD+RW).

The present invention also provides polypeptide fragments or peptidescomprising an epitope-bearing portion of a mouse Zcytor16 polypeptidedescribed herein. Such fragments or peptides may comprise an“immunogenic epitope,” which is a part of a protein that elicits anantibody response when the entire protein is used as an immunogen.Immunogenic epitope-bearing peptides can be identified using standardmethods (see, for example, Geysen et al., Proc. Nat'l Acad. Sci. USA81:3998 (1983)).

In contrast, polypeptide fragments or peptides may comprise an“antigenic epitope,” which is a region of a protein molecule to which anantibody can specifically bind. Certain epitopes consist of a linear orcontiguous stretch of amino acids, and the antigenicity of such anepitope is not disrupted by denaturing agents. It is known in the artthat relatively short synthetic peptides that can mimic epitopes of aprotein can be used to stimulate the production of antibodies againstthe protein (see, for example, Sutcliffe et al., Science 219:660(1983)). Accordingly, antigenic epitope-bearing peptides andpolypeptides of the present invention are useful to raise antibodiesthat bind with the polypeptides described herein.

Antigenic epitope-bearing peptides and polypeptides can contain at leastfour to ten amino acids, at least ten to fifteen amino acids, or about15 to about 30 amino acids of an amino acid sequence disclosed herein.Such epitope-bearing peptides and polypeptides can be produced byfragmenting a mouse Zcytor16 polypeptide, or by chemical peptidesynthesis, as described herein. Moreover, epitopes can be selected byphage display of random peptide libraries (see, for example, Lane andStephen, Curr. Opin. Immunol. 5:268 (1993), and Cortese et al., Curr.Opin. Biotechnol. 7:616 (1996)). Standard methods for identifyingepitopes and producing antibodies from small peptides that comprise anepitope are described, for example, by Mole, “Epitope Mapping,” inMethods in Molecular Biology, Vol. 10, Manson (ed.), pages 105-116 (TheHumana Press, Inc. 1992), Price, “Production and Characterization ofSynthetic Peptide-Derived Antibodies,” in Monoclonal Antibodies:Production, Engineering, and Clinical Application, Ritter and Ladyman(eds.), pages 60-84 (Cambridge University Press 1995), and Coligan etal. (eds.), Current Protocols in Immunology, pages 9.3.1-9.3.5 and pages9.4.1-9.4.11 (John Wiley & Sons 1997).

6. Production of Mouse Zcytor16 Polypeptides

The polypeptides of the present invention, including full-lengthpolypeptides; soluble monomeric, homodimeric, heterodimeric andmultimeric receptors; full-length receptors; receptor fragments (e.g.ligand-binding fragments), functional fragments, and fusion proteins,can be produced in recombinant host cells following conventionaltechniques. To express a mouse Zcytor16 gene, a nucleic acid moleculeencoding the polypeptide must be operably linked to regulatory sequencesthat control transcriptional expression in an expression vector andthen, introduced into a host cell. In addition to transcriptionalregulatory sequences, such as promoters and enhancers, expressionvectors can include translational regulatory sequences and a marker genewhich is suitable for selection of cells that carry the expressionvector.

Expression vectors that are suitable for production of a foreign proteinin eukaryotic cells typically contain (1) prokaryotic DNA elementscoding for a bacterial replication origin and an antibiotic resistancemarker to provide for the growth and selection of the expression vectorin a bacterial host; (2) eukaryotic DNA elements that control initiationof transcription, such as a promoter; and (3) DNA elements that controlthe processing of transcripts, such as a transcriptiontermination/polyadenylation sequence. As discussed above, expressionvectors can also include nucleotide sequences encoding a secretorysequence that directs the heterologous polypeptide into the secretorypathway of a host cell. For example, a mouse Zcytor16 expression vectormay comprise a mouse Zcytor16 gene and a secretory sequence derived fromany secreted gene.

Mouse Zcytor16 proteins of the present invention may be expressed inmammalian cells. Examples of suitable mammalian host cells includeAfrican green monkey kidney cells (Vero; ATCC CRL 1587), human embryonickidney cells (293-HEK; ATCC CRL 1573), baby hamster kidney cells(BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314), canine kidney cells(MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1; ATCC CCL61;CHO DG44 (Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986)), ratpituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rathepatoma cells (H-4-II-E; ATCC CRL 1548) SV40-transformed monkey kidneycells (COS-1; ATCC CRL 1650) and murine embryonic cells (NIH-3T3; ATCCCRL 1658).

For a mammalian host, the transcriptional and translational regulatorysignals may be derived from viral sources, such as adenovirus, bovinepapilloma virus, simian virus, or the like, in which the regulatorysignals are associated with a particular gene which has a high level ofexpression. Suitable transcriptional and translational regulatorysequences also can be obtained from mammalian genes, such as actin,collagen, myosin, and metallothionein genes.

Transcriptional regulatory sequences include a promoter regionsufficient to direct the initiation of RNA synthesis. Suitableeukaryotic promoters include the promoter of the mouse metallothionein Igene (Hamer et al., J. Molec. Appl. Genet. 1:273 (1982)), the TKpromoter of Herpes virus (McKnight, Cell 31:355 (1982)), the SV40 earlypromoter (Benoist et al., Nature 290:304 (1981)), the Rous sarcoma viruspromoter (Gorman et al., Proc. Nat'l Acad. Sci. USA 79:6777 (1982)), thecytomegalovirus promoter (Foecking et al., Gene 45:101 (1980)), and themouse mammary tumor virus promoter (see, generally, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163-181 (John Wiley & Sons, Inc. 1996)).

Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNApolymerase promoter, can be used to control mouse Zcytor16 geneexpression in mammalian cells if the prokaryotic promoter is regulatedby a eukaryotic promoter (Zhou et al., Mol. Cell. Biol. 10:4529 (1990),and Kaufman et al., Nucl. Acids Res. 19:4485 (1991)).

In certain embodiments, a DNA sequence encoding a mouse Zcytor16monomeric or homodimeric soluble receptor polypeptide, or a DNA sequenceencoding an additional subunit of a heterodimeric or multimeric mouseZcytor16 soluble receptor, e.g., CRF2-4 or IL10R, polypeptide isoperably linked to other genetic elements required for its expression,generally including a transcription promoter and terminator, within anexpression vector. The vector will also commonly contain one or moreselectable markers and one or more origins of replication, althoughthose skilled in the art will recognize that within certain systemsselectable markers may be provided on separate vectors, and replicationof the exogenous DNA may be provided by integration into the host cellgenome. Selection of promoters, terminators, selectable markers, vectorsand other elements is a matter of routine design within the level ofordinary skill in the art. Many such elements are described in theliterature and are available through commercial suppliers. Multiplecomponents of a soluble receptor complex can be co-transfected onindividual expression vectors or be contained in a single expressionvector. Such techniques of expressing multiple components of proteincomplexes are well known in the art.

An expression vector can be introduced into host cells using a varietyof standard techniques including calcium phosphate transfection,liposome-mediated transfection, microprojectile-mediated delivery,electroporation, and the like. The transfected cells can be selected andpropagated to provide recombinant host cells that comprise theexpression vector stably integrated in the host cell genome. Techniquesfor introducing vectors into eukaryotic cells and techniques forselecting such stable transformants using a dominant selectable markerare described, for example, by Ausubel (1995) and by Murray (ed.), GeneTransfer and Expression Protocols (Humana Press 1991).

For example, one suitable selectable marker is a gene that providesresistance to the antibiotic neomycin. In this case, selection iscarried out in the presence of a neomycin-type drug, such as G-418 orthe like. Selection systems can also be used to increase the expressionlevel of the gene of interest, a process referred to as “amplification.”Amplification is carried out by culturing transfectants in the presenceof a low level of the selective agent and then increasing the amount ofselective agent to select for cells that produce high levels of theproducts of the introduced genes. A suitable amplifiable selectablemarker is dihydrofolate reductase, which confers resistance tomethotrexate. Other drug resistance genes (e.g., hygromycin resistance,multi-drug resistance, puromycin acetyltransferase) can also be used.Alternatively, markers that introduce an altered phenotype, such asgreen fluorescent protein, or cell surface proteins such as CD4, CD8,Class I MHC, placental alkaline phosphatase may be used to sorttransfected cells from untransfected cells by such means as FACS sortingor magnetic bead separation technology.

Mouse Zcytor16 polypeptides can also be produced by cultured mammaliancells using a viral delivery system. Exemplary viruses for this purposeinclude adenovirus, retroviruses, herpesvirus, vaccinia virus andadeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus,is currently the best studied gene transfer vector for delivery ofheterologous nucleic acid (for a review, see Becker et al., Meth. CellBiol. 43:161 (1994), and Douglas and Curiel, Science & Medicine 4:44(1997)). Advantages of the adenovirus system include the accommodationof relatively large DNA inserts, the ability to grow to high-titer, theability to infect a broad range of mammalian cell types, and flexibilitythat allows use with a large number of available vectors containingdifferent promoters.

By deleting portions of the adenovirus genome, larger inserts (up to 7kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. An option is to delete theessential E1 gene from the viral vector, which results in the inabilityto replicate unless the E1 gene is provided by the host cell. Adenovirusvector-infected human 293 cells (ATCC Nos. CRL-1573, 45504, 45505), forexample, can be grown as adherent cells or in suspension culture atrelatively high cell density to produce significant amounts of protein(see Garnier et al., Cytotechnol. 15:145 (1994)).

Mouse Zcytor16 can also be expressed in other higher eukaryotic cells,such as avian, fungal, insect, yeast, or plant cells. The baculovirussystem provides an efficient means to introduce cloned mouse Zcytor16genes into insect cells. Suitable expression vectors are based upon theAutographa californica multiple nuclear polyhedrosis virus (AcMNPV), andcontain well-known promoters such as Drosophila heat shock protein (hsp)70 promoter, Autographa californica nuclear polyhedrosis virusimmediate-early gene promoter (ie-1) and the delayed early 39K promoter,baculovirus p10 promoter, and the Drosophila metallothionein promoter. Asecond method of making recombinant baculovirus utilizes atransposon-based system described by Luckow (Luckow, et al., J. Virol.67:4566 (1993)). This system, which utilizes transfer vectors, is soldin the BAC-to-BAC kit (Life Technologies, Rockville, Md.). This systemutilizes a transfer vector, PFASTBAC (Life Technologies) containing aTn7 transposon to move the DNA encoding the mouse Zcytor16 polypeptideinto a baculovirus genome maintained in E. coli as a large plasmidcalled a “bacmid.” See, Hill-Perkins and Possee, J. Gen. Virol. 71:971(1990), Bonning, et al., J. Gen. Virol. 75:1551 (1994), and Chazenbalk,and Rapoport, J. Biol. Chem. 270:1543 (1995). In addition, transfervectors can include an in-frame fusion with DNA encoding an epitope tagat the C- or N-terminus of the expressed mouse Zcytor16 polypeptide, forexample, a Glu-Glu epitope tag (Grussenmeyer et al., Proc. Nat'l Acad.Sci. 82:7952 (1985)). Using a technique known in the art, a transfervector containing a mouse Zcytor16 gene is transformed into E. coli, andscreened for bacmids which contain an interrupted lacZ gene indicativeof recombinant baculovirus. The bacmid DNA containing the recombinantbaculovirus genome is then isolated using common techniques.

The illustrative PFASTBAC vector can be modified to a considerabledegree. For example, the polyhedrin promoter can be removed andsubstituted with the baculovirus basic protein promoter (also known asPcor, p6.9 or MP promoter) which is expressed earlier in the baculovirusinfection, and has been shown to be advantageous for expressing secretedproteins (see, for example, Hill-Perkins and Possee, J. Gen. Virol.71:971 (1990), Bonning, et al., J. Gen. Virol. 75:1551 (1994), andChazenbalk and Rapoport, J. Biol. Chem. 270:1543 (1995). In suchtransfer vector constructs, a short or long version of the basic proteinpromoter can be used. Moreover, transfer vectors can be constructedwhich replace the native mouse Zcytor16 secretory signal sequences withsecretory signal sequences derived from insect proteins. For example, asecretory signal sequence from Ecdysteroid Glucosyltransferase (EGT),honey bee Melittin (Invitrogen Corporation; Carlsbad, Calif.), orbaculovirus gp67 (PharMingen: San Diego, Calif.) can be used inconstructs to replace the native mouse Zcytor16 secretory signalsequence.

The recombinant virus or bacmid is used to transfect host cells.Suitable insect host cells include cell lines derived from IPLB-Sf-21, aSpodoptera frugiperda pupal ovarian cell line, such as Sf9 (ATCC CRL1711), Sf21AE, and Sf21 (Invitrogen Corporation; San Diego, Calif.), aswell as Drosophila Schneider-2 cells, and the HIGH FIVEO cell line(Invitrogen) derived from Trichoplusia ni (U.S. Pat. No. 5,300,435).Commercially available serum-free media can be used to grow and tomaintain the cells. Suitable media are Sf900 II™ (Life Technologies) orESF 921™ (Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRHBiosciences, Lenexa, Kans.) or Express FiveO™ (Life Technologies) forthe T. ni cells. When recombinant virus is used, the cells are typicallygrown up from an inoculation density of approximately 2-5×10⁵ cells to adensity of 1-2×10⁶ cells at which time a recombinant viral stock isadded at a multiplicity of infection (MOI) of 0.1 to 10, more typicallynear 3.

Established techniques for producing recombinant proteins in baculovirussystems are provided by Bailey et al., “Manipulation of BaculovirusVectors,” in Methods in Molecular Biology, Volume 7: Gene Transfer andExpression Protocols, Murray (ed.), pages 147-168 (The Humana Press,Inc. 1991), by Patel et al., “The baculovirus expression system,” in DNACloning 2: Expression Systems,: 2nd Edition, Glover et al. (eds.), pages205-244 (Oxford University Press 1995), by Ausubel (1995) at pages 16-37to 16-57, by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc. 1995), and by Lucknow, “Insect Cell ExpressionTechnology,” in Protein Engineering: Principles and Practice, Cleland etal. (eds.), pages 183-218 (John Wiley & Sons, Inc. 1996).

Fungal cells, including yeast cells, can also be used to express thegenes described herein. Yeast species of particular interest in thisregard include Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Suitable promoters for expression in yeast includepromoters from GAL1 (galactose), PGK (phosphoglycerate kinase), ADH(alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinoldehydrogenase), and the like. Many yeast cloning vectors have beendesigned and are readily available. These vectors include YIp-basedvectors, such as YIp5, YRp vectors, such as YRp17, YEp vectors such asYEp13 and YCp vectors, such as YCp19. Methods for transforming S.cerevisiae cells with exogenous DNA and producing recombinantpolypeptides therefrom are disclosed by, for example, Kawasaki, U.S.Pat. No. 4,599,311, Kawasaki et al., U.S. Pat. No. 4,931,373, Brake,U.S. Pat. No. 4,870,008, Welch et al., U.S. Pat. No. 5,037,743, andMurray et al., U.S. Pat. No. 4,845,075. Transformed cells are selectedby phenotype determined by the selectable marker, commonly drugresistance or the ability to grow in the absence of a particularnutrient (e.g., leucine). A suitable vector system for use inSaccharomyces cerevisiae is the POT1 vector system disclosed by Kawasakiet al. (U.S. Pat. No. 4,931,373), which allows transformed cells to beselected by growth in glucose-containing media. Additional suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311, Kingsman etal., U.S. Pat. No. 4,615,974, and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446,5,063,154, 5,139,936, and 4,661,454.

Transformation systems for other yeasts, including Hansenula polymorpha,Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis,Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichiaguillermondii and Candida maltosa are known, in the art. See, forexample Gleeson et al. J. Gen. Microbiol. 132:3459 (1986), and Cregg,U.S. Pat. No. 4,882,279. Aspergillus cells may be utilized according tothe methods of McKnight et al., U.S. Pat. No. 4,935,349. Methods fortransforming Acremonium chrysogenum are disclosed by Sumino et al., U.S.Pat. No. 5,162,228. Methods for transforming Neurospora are disclosed byLambowitz, U.S. Pat. No. 4,486,533.

For example, the use of Pichia methanolica as host for the production ofrecombinant proteins is disclosed by Raymond, U.S. Pat. No. 5,716,808,Raymond, U.S. Pat. No. 5,736,383, Raymond et al., Yeast 14:11-23 (1998),and in international publication Nos. WO 97/17450, WO 97/17451, WO98/02536, and WO 98/02565. DNA molecules for use in transforming P.methanolica will commonly be prepared as double-stranded, circularplasmids, which are preferably linearized prior to transformation. Forpolypeptide production in P. methanolica, the promoter and terminator inthe plasmid can be that of a P. methanolica gene, such as a P.methanolica alcohol utilization gene (AUG1 or AUG2). Other usefulpromoters include those of the dihydroxyacetone synthase (DHAS), formatedehydrogenase (FMD), and catalase (CAT) genes. To facilitate integrationof the DNA into the host chromosome, it is preferred to have the entireexpression segment of the plasmid flanked at both ends by host DNAsequences. A suitable selectable marker for use in Pichia methanolica isa P. methanolica ADE2 gene, which encodesphosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), andwhich allows ade2 host cells to grow in the absence of adenine. Forlarge-scale, industrial processes where it is desirable to minimize theuse of methanol, host cells can be used in which both methanolutilization genes (AUG1 and AUG2) are deleted. For production ofsecreted proteins, host cells can be deficient in vacuolar proteasegenes (PEP4 and PRB1). Electroporation is used to facilitate theintroduction of a plasmid containing DNA encoding a polypeptide ofinterest into P. methanolica cells. P. methanolica cells can betransformed by electroporation using an exponentially decaying, pulsedelectric field having a field strength of from 2.5 to 4.5 kV/cm,preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40milliseconds, most preferably about 20 milliseconds.

Expression vectors can also be introduced into plant protoplasts, intactplant tissues, or isolated plant cells. Methods for introducingexpression vectors into plant tissue include the direct infection orco-cultivation of plant tissue with Agrobacterium tumefaciens,microprojectile-mediated delivery, DNA injection, electroporation, andthe like. See, for example, Horsch et al., Science 227:1229 (1985),Klein et al., Biotechnology 10:268 (1992), and Miki et al., “Proceduresfor Introducing Foreign DNA into Plants,” in Methods in Plant MolecularBiology and Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press,1993).

Alternatively, mouse Zcytor16 genes can be expressed in prokaryotic hostcells. Suitable promoters that can be used to express mouse Zcytor16polypeptides in a prokaryotic host are well-known to those of skill inthe art and include promoters capable of recognizing the T4, T3, Sp6 andT7 polymerases, the P_(R) and P_(L) promoters of bacteriophage lambda,the trp, recA, heat shock, lacUV5, tac, lpp-lacSpr, phoA, and lacZpromoters of E. coli, promoters of B. subtilis, the promoters of thebacteriophages of Bacillus, Streptomyces promoters, the int promoter ofbacteriophage lambda, the bla promoter of pBR322, and the CAT promoterof the chloramphenicol acetyl transferase gene. Prokaryotic promotershave been reviewed by Glick, J. Ind. Microbiol. 1:277 (1987), Watson etal., Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins 1987), andby Ausubel et al. (1995).

Suitable prokaryotic hosts include E. coli and Bacillus subtilus.Suitable strains of E. coli include BL21(DE3), BL21(DE3)pLysS,BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF′, DH5IMCR, DH10B, DH10B/p3,DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089,CSH18, ER1451, and ER1647 (see, for example, Brown (ed.), MolecularBiology Labfax (Academic Press 1991)). Suitable strains of Bacillussubtilus include BR151, YB886, M1119, MI120, and B170 (see, for example,Hardy, “Bacillus Cloning Methods,” in DNA Cloning: A Practical Approach,Glover (ed.) (IRL Press 1985)).

When expressing a mouse Zcytor16 polypeptide in bacteria such as E.coli, the polypeptide may be retained in the cytoplasm, typically asinsoluble granules, or may be directed to the periplasmic space by abacterial secretion sequence. In the former case, the cells are lysed,and the granules are recovered and denatured using, for example,guanidine isothiocyanate or urea. The denatured polypeptide can then berefolded and dimerized by diluting the denaturant, such as by dialysisagainst a solution, of urea and a combination of reduced and oxidizedglutathione, followed by dialysis against a buffered saline solution. Inthe latter case, the polypeptide can be recovered from the periplasmicspace in a soluble and functional form by disrupting the cells (by, forexample, sonication or osmotic shock) to release the contents of theperiplasmic space and recovering the protein, thereby obviating the needfor denaturation and refolding.

Methods for expressing proteins in prokaryotic hosts are well-known tothose of skill in the art (see, for example, Williams et al.,“Expression of foreign proteins in E. coli using plasmid vectors andpurification of specific polyclonal antibodies,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (OxfordUniversity Press 1995), Ward et al., “Genetic Manipulation andExpression of Antibodies,” in Monoclonal Antibodies: Principles andApplications, page 137 (Wiley-Liss, Inc. 1995), and Georgiou,“Expression of Proteins in Bacteria,” in Protein Engineering: Principlesand Practice, Cleland et al. (eds.), page 101 (John Wiley & Sons, Inc.1996)).

Standard methods for introducing expression vectors into bacterial,yeast, insect, and plant cells are provided, for example, by Ausubel(1995).

General methods for expressing and recovering foreign protein producedby a mammalian cell system are provided by, for example, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163 (Wiley-Liss, Inc. 1996). Standard techniques for recoveringprotein produced by a bacterial system is provided by, for example,Grisshammer et al., “Purification of over-produced proteins from E. colicells,” in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al.(eds.), pages 59-92 (Oxford University Press 1995). Established methodsfor isolating recombinant proteins from a baculovirus system aredescribed by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc. 1995).

As an alternative, polypeptides of the present invention can besynthesized by exclusive solid phase synthesis, partial solid phasemethods, fragment condensation or classical solution synthesis. Thesesynthesis methods are well-known to those of skill in the art (see, forexample, Merrifield, J. Am. Chem. Soc. 85:2149 (1963), Stewart et al.,“Solid Phase Peptide Synthesis” (2nd Edition), (Pierce Chemical Co.1984), Bayer and Rapp, Chem. Pept. Prot. 3:3 (1986), Atherton et al.,Solid Phase Peptide Synthesis: A Practical Approach (IRL Press 1989),Fields and Colowick, “Solid-Phase Peptide Synthesis,” Methods inEnzymology Volume 289 (Academic Press 1997), and Lloyd-Williams et al.,Chemical Approaches to the Synthesis of Peptides and Proteins (CRCPress, Inc. 1997)). Variations in total chemical synthesis strategies,such as “native chemical ligation” and “expressed protein ligation” arealso standard (see, for example, Dawson et al., Science 266:776 (1994),Hackeng et al., Proc. Nat'l Acad. Sci. USA 94:7845 (1997), Dawson,Methods Enzymol. 287: 34 (1997), Muir et al, Proc. Nat'l Acad. Sci. USA95:6705 (1998), and Severinov and Muir, J. Biol. Chem. 273:16205(1998)).

Peptides and polypeptides of the present invention comprise at leastsix, at least nine, or at least 15 contiguous amino acid residues of SEQID NO:38 or SEQ ID NO:48. As an illustration, polypeptides can compriseat least six, at least nine, or at least 15 contiguous amino acidresidues of any of the following amino acid sequences of SEQ ID NO:38 orSEQ ID NO:48: amino acid residues amino acid residues 1 to 230, aminoacid residues 24 to 230, or 27 to 230, and amino acid residues 27 to126, amino acid residues 131 to 230, or other fragments disclosedherein. Within certain embodiments of the invention, the polypeptidescomprise 20, 30, 40, 50, 100, or more contiguous residues of these aminoacid sequences. Nucleic acid molecules encoding such peptides andpolypeptides are useful as polymerase chain reaction primers and probes.

Moreover, mouse Zcytor16 polypeptides can be expressed as monomers,homodimers, heterodimers, or multimers within higher eukaryotic cells.Such cells can be used to produce mouse Zcytor16 monomeric, homodimeric,heterodimeric and multimeric receptor polypeptides that comprise atleast one mouse Zcytor16 polypeptide (“mouse Zcytor16-comprisingreceptors” or “mouse Zcytor16-comprising receptor polypeptides”), or canbe used as assay cells in screening systems. Within one aspect of thepresent invention, a polypeptide of the present invention comprising themouse Zcytor16 extracellular domain is produced by a cultured cell, andthe cell is used to screen for ligands for the receptor, including thenatural ligand, IL-TIF, as well as agonists and antagonists of thenatural ligand. To summarize this approach, a cDNA or gene encoding thereceptor is combined with other genetic elements required for itsexpression (e.g., a transcription promoter), and the resultingexpression vector is inserted into a host cell. Cells that express theDNA and produce functional receptor are selected and used within avariety of screening systems. Each component of the monomeric,homodimeric, heterodimeric and multimeric receptor complex can beexpressed in the same cell. Moreover, the components of the monomeric,homodimeric, heterodimeric and multimeric receptor complex can also befused to a transmembrane domain or other membrane fusion moiety to allowcomplex assembly and screening of transfectants as described above.

Mammalian cells suitable for use in expressing mouse Zcytor16 receptorsand transducing a receptor-mediated signal include cells that expressother receptor subunits that may form a functional complex with mouseZcytor16. These subunits may include those of the interferon receptorfamily or of other class II or class I cytokine receptors, e.g., CRF2-4(Genbank Accession No. Z17227), IL-10R (Genbank Accession No.s U00672and NM_(—)001558), zcytor11 (U.S. Pat. No. 5,965,704), zcytor7 (U.S.Pat. No. 5,945,511), and IL-9R. It is also preferred to use a cell fromthe same species as the receptor to be expressed. Within a preferredembodiment, the cell is dependent upon an exogenously suppliedhematopoietic growth factor for its proliferation. Preferred cell linesof this type are the human TF-1 cell line (ATCC number CRL-2003) and theAML-193 cell line (ATCC number CRL-9589), which are GM-CSF-dependenthuman leukemic cell lines and BaF3 (Palacios and Steinmetz, Cell 41:727-734, (1985)) which is an IL-3 dependent murine pre-B cell line.Other cell lines include BHK, COS-1 and CHO cells.

Suitable host cells can be engineered to produce the necessary receptorsubunits or other cellular component needed for the desired cellularresponse. This approach is advantageous because cell lines can beengineered to express receptor subunits from any species, therebyovercoming potential limitations arising from species specificity.Species orthologs of the mouse receptor cDNA can be cloned and usedwithin cell lines from the same species, such as a mouse cDNA in theBaF3 cell line. Cell lines that are dependent upon one hematopoieticgrowth factor, such as GM-CSF or IL-3, can thus be engineered to becomedependent upon another cytokine that acts through the mouse Zcytor16receptor, such as IL-TIF.

Cells expressing functional receptor are used within screening assays. Avariety of suitable assays are known in the art. These assays are basedon the detection of a biological response in a target cell. One suchassay is a cell proliferation assay. Cells are cultured in the presenceor absence of a test compound, and cell proliferation is detected by,for example, measuring incorporation of tritiated thymidine or bycolorimetric assay based on the metabolic breakdown of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)(Mosman, J. Immunol. Meth. 65: 55-63, (1983)). An alternative assayformat uses cells that are further engineered to express a reportergene. The reporter gene is linked to a promoter element that isresponsive to the receptor-linked pathway, and the assay detectsactivation of transcription of the reporter gene. A preferred promoterelement in this regard is a serum response element, or SRE. See, e.g.,Shaw et al., Cell 56:563-572, (1989). A preferred such reporter gene isa luciferase gene (de Wet et al., Mol. Cell. Biol. 7:725, (1987)).Expression of the luciferase gene is detected by luminescence usingmethods known in the art (e.g., Baumgartner et al., J. Biol. Chem.269:29094-29101, (1994); Schenborn and Goiffin, Promega _(—) Notes41:11, 1993). Luciferase activity assay kits are commercially availablefrom, for example, Promega Corp., Madison, Wis. Target cell lines ofthis type can be used to screen libraries of chemicals, cell-conditionedculture media, fungal broths, soil samples, water samples, and the like.For example, a bank of cell-conditioned media samples can be assayed ona target cell to identify cells that produce ligand. Positive cells arethen used to produce a cDNA library in a mammalian expression vector,which is divided into pools, transfected into host cells, and expressed.Media samples from the transfected cells are then assayed, withsubsequent division of pools, re-transfection, subculturing, andre-assay of positive cells to isolate a cloned cDNA encoding the ligand.

A natural ligand for the mouse Zcytor16 receptor can also be identifiedby mutagenizing a cell line expressing the full-length receptor orreceptor fusion (e.g, comprising the mouse Zcytor16 extracellular domianfused to the transmembrane and signaling domain of another cytokinereceptor) and culturing it under conditions that select for autocrinegrowth. See WIPO publication WO 95/21930. Within a typical procedure,IL-3 dependent BaF3 cells expressing mouse Zcytor16 and the necessaryadditional subunits are mutagenized, such as with2-ethylmethanesulfonate (EMS). The cells are then allowed to recover inthe presence of IL-3, then transferred to a culture medium lacking IL-3and IL-4. Surviving cells are screened for the production of a Zcytor16ligand (e.g., IL-TIF), such as by adding soluble receptor to the culturemedium or by assaying conditioned media on wild-type BaF3 cells and BaF3cells expressing the receptor. Using this method, cells and tissuesexpressing IL-TIF can be identified. Such methods can be employed todetect IL-TIF from different species, e.g., human, and as such cells,cancers, and tissues expressing human IL-TIF can be identified using themouse Zcytor16 polypeptides of the present invention.

Moreover several IL-TIF responsive cell lines are known (Dumontier etal., J. Immunol. 164:1814-1819, 2000; Dumoutier, L. et al., Proc. Nat'l.Acad. Sci. 97:10144-10149, 2000; Xie M H et al., J. Biol. Chem. 275:31335-31339, 2000; Kotenko S V et al., JBC in press), as well as thosethat express the IL-TIF receptor subunit zcytor11. For example, thefollowing cells are responsive to IL-TIF: TK-10 (Xie M H et al., supra.)(human renal carcinoma); SW480 (ATCC No. CCL-228) (human colonadenocarcinoma); HepG2 (ATCC No. HB-8065) (human hepatoma); PC12 (ATCCNo. CRL-1721) (murine neuronal cell model; rat pheochromocytoma); andMES13 (ATCC No. CRL-1927) (murine kidney mesangial cell line). Inaddition, some cell lines express zcytor11 (IL-TIF receptor) are alsocandidates for responsive cell lines to IL-TIF: A549 (ATCC No. CCL-185)(human lung carcinoma); G-361 (ATCC No. CRL-1424) (human melanoma); andCaki-1 (ATCC No. HTB-46) (human renal carcinoma). These cells can beused in assays to assess the functionality of mouse Zcytor16 as anIL-TIF antagonist or anti-inflammatory factor, even in heterologoussystems, such as human.

An additional screening approach provided by the present inventionincludes the use of hybrid receptor polypeptides. These hybridpolypeptides fall into two general classes. Within the first class, theintracellular domain of mouse Zcytor16, is joined to the ligand-bindingdomain of a second receptor. It is preferred that the second receptor bea hematopoietic cytokine receptor, such as mp1 receptor (Souyri et al.,Cell .63: 1137-1147, (1990). The hybrid receptor will further comprise atransmembrane domain, which may be derived from either receptor. A DNAconstruct encoding the hybrid receptor is then inserted into a hostcell. Cells expressing the hybrid receptor are cultured in the presenceof a ligand for the binding domain (e.g., TPO in the case the mp1receptor extracellular domain is used) and assayed for a response. Thissystem provides a means for analyzing signal transduction mediated bymouse Zcytor16 while using readily available ligands. This system canalso be used to determine if particular cell lines are capable ofresponding to signals transduced by mouse Zcytor16 monomeric,homodimeric, heterodimeric and multimeric receptors of the presentinvention.

A second class of hybrid receptor polypeptides comprise theextracellular (ligand-binding) domain of mouse Zcytor16 (approximatelyresidues 24 to 230, or 27 to 230 of SEQ ID NO:38 or SEQ ID NO:48) withan intracellular domain of a second receptor, preferably a hematopoieticcytokine receptor, and a transmembrane domain. Hybrid zacytor11monomers, homodimers, heterodimers and multimers of the presentinvention receptors of this second class are expressed in cells known tobe capable of responding to signals transduced by the second receptor.Together, these two classes of hybrid receptors enable theidentification of a responsive cell type for the development of an assayfor detecting IL-TIF. Moreover, such cells can be used in the presenceof IL-TIF to assay the soluble receptor antagonists of the presentinvention in a competition-type assay. In such assay, a decrease in theproliferation or signal transduction activity of IL-TIF in the presenceof a soluble receptor of the present invention demonstrates antagonisticactivity. Moreover IL-TIF-soluble receptor binding assays can also beused to assess whether a soluble receptor antagonizes IL-TIF activity.

7. Production of Mouse Zcytor16 Fusion Proteins and Conjugates

One general class of mouse Zcytor16 analogs are variants having an aminoacid sequence that is a mutation of the amino acid sequence disclosedherein. Another general class of mouse Zcytor16 analogs is provided byanti-idiotype antibodies, and fragments thereof, as described below.Moreover, recombinant antibodies comprising anti-idiotype variabledomains can be used as analogs (see, for example, Monfardini et al.,Proc. Assoc. Am. Physicians 108:420 (1996)). Since the variable domainsof anti-idiotype mouse Zcytor16 antibodies mimic mouse Zcytor16, thesedomains can provide mouse or human Zcytor16 binding activity, e.g., toIL-TIF. Methods of producing anti-idiotypic catalytic antibodies areknown to those of skill in the art (see, for example, Joron et al., Ann.N Y Acad. Sci. 672:216 (1992), Friboulet et al., Appl. Biochem.Biotechnol. 47:229 (1994), and Avalle et al., Ann. N Y Acad. Sci.864:118 (1998)).

Another approach to identifying Zcytor16 analogs is provided by the useof combinatorial libraries. Methods for constructing and screening phagedisplay and other combinatorial libraries are provided, for example, byKay et al., Phage Display of Peptides and Proteins (Academic Press1996), Verdine, U.S. Pat. No. 5,783,384, Kay, et. al., U.S. Pat. No.5,747,334, and Kauffman et al., U.S. Pat. No. 5,723,323.

Mouse Zcytor16 polypeptides have both in vivo and in vitro uses. As anillustration, a soluble form of mouse Zcytor16 can be added to cellculture medium to inhibit the effects of the Zcytor16 ligand produced bythe cultured cells.

Fusion proteins of mouse Zcytor16 can be used to express mouse Zcytor16in a recombinant host, and to isolate the produced mouse Zcytor16. Asdescribed below, particular mouse Zcytor16 fusion proteins also haveuses in diagnosis and therapy. One type of fusion protein comprises apeptide that guides a mouse Zcytor16 polypeptide from a recombinant hostcell. To direct a mouse Zcytor16 polypeptide into the secretory pathwayof a eukaryotic host cell, a secretory signal sequence (also known as asignal peptide, a leader sequence, prepro sequence or pre sequence) isprovided in the mouse Zcytor16 expression vector. While the secretorysignal sequence may be derived from mouse Zcytor16, a suitable signalsequence may also be derived from another secreted protein orsynthesized de novo. The secretory signal sequence is operably linked toa mouse Zcytor16-encoding sequence such that the two sequences arejoined in the correct reading frame and positioned to direct the newlysynthesized polypeptide into the secretory pathway of the host cell.Secretory signal sequences are commonly positioned 5′ to the nucleotidesequence encoding the polypeptide of interest, although certainsecretory signal sequences may be positioned elsewhere in the nucleotidesequence of interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743;Holland et al., U.S. Pat. No. 5,143,830).

Although the secretory signal sequence of mouse Zcytor16 or anotherprotein produced by mammalian cells (e.g., tissue-type plasminogenactivator signal sequence, as described, for example, in U.S. Pat. No.5,641,655) is useful for expression of mouse Zcytor16 in recombinantmammalian hosts, a yeast signal sequence is preferred for expression inyeast cells. Examples of suitable yeast signal sequences are thosederived from yeast mating phermone cc-factor (encoded by the MFal gene),invertase (encoded by the SUC2 gene), or acid phosphatase (encoded bythe PHO5 gene). See, for example, Romanos et al., “Expression of ClonedGenes in Yeast,” in DNA Cloning 2: A Practical Approach, 2^(nd) Edition,Glover and Hames (eds.), pages 123-167 (Oxford University Press 1995).

Mouse Zcytor16 monomeric, homodimeric, heterodimeric and multimericreceptor polypeptides can be prepared by expressing a truncated DNAencoding the extracellular domain, for example, a polypeptide whichcontains 24 to 230, or 27 to 230 of SEQ ID NO:38 or SEQ ID NO:48, or thecorresponding region of a non-mouse receptor. It is preferred that theextracellular domain polypeptides be prepared in a form substantiallyfree of transmembrane and intracellular polypeptide segments. To directthe export of the receptor domain from the host cell, the receptor DNAis linked to a second DNA segment encoding a secretory peptide, such asa t-PA secretory peptide. To facilitate purification of the secretedreceptor domain, a C-terminal extension, such as a poly-histidine tag,substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-1210,(1988); available from Eastman Kodak Co., New Haven, Conn.) or anotherpolypeptide or protein for which an antibody or other specific bindingagent is available, can be fused to the receptor polypeptide. Moreover,heterodimeric and multimeric non-mouse Zcytor16 subunit extracellularcytokine binding domains are a also prepared as above.

In an alternative approach, a receptor extracellular domain of mouseZcytor16 or other class I or II cytokine receptor component can beexpressed as a fusion with immunoglobulin heavy chain constant regions,typically an F_(c) fragment, which contains two constant region domainsand a hinge region but lacks the variable region (See, Sledziewski, A Zet al., U.S. Pat. No. 6,018,026 and 5,750,375). The soluble mouseZcytor16, soluble mouse Zcytor16/CRF2-4 heterodimers, and monomeric,homodimeric, heterodimeric and multimeric polypeptides of the presentinvention include such fusions. Such fusions are typically secreted asmultimeric molecules wherein the Fc portions are disulfide bonded toeach other and two receptor polypeptides are arrayed in closed proximityto each other. Fusions of this type can be used to affinity purify thecognate ligand from solution, as an in vitro assay tool, to blocksignals in vitro by specifically titrating out ligand, and asantagonists in vivo by administering them parenterally to bindcirculating ligand and clear it from the circulation. To purify ligand,a mouse Zcytor16-Ig chimera is added to a sample containing the ligand(e.g., cell-conditioned culture media or tissue extracts) underconditions that facilitate receptor-ligand binding (typicallynear-physiological temperature, pH, and ionic strength). Thechimera-ligand complex is then separated by the mixture using protein A,which is immobilized on a solid support (e.g., insoluble resin beads).The ligand is then eluted using conventional chemical techniques, suchas with a salt or pH gradient. In the alternative, the chimera itselfcan be bound to a solid support, with binding and elution carried out asabove. The chimeras may be used in vivo to regulate inflammatoryresponses including acute phase responses such as serum amyloid A (SAA),C-reactive protein (CRP), and the like. Chimeras with high bindingaffinity are administered parenterally (e.g., by intramuscular,subcutaneous or intravenous injection). Circulating molecules bindligand and are cleared from circulation by normal physiologicalprocesses. For use in assays, the chimeras are bound to a support viathe F_(c) region and used in an ELISA format. Moreover, susch methodscan be applied using the polypeptides of the present invention toisolate mouse IL-TIF, as well as an orthologous ligand, e.g., humanIL-TIF.

The present invention further provides a variety of other polypeptidefusions and related multimeric proteins comprising one or morepolypeptide fusions. For example, a soluble mouse Zcytor16 receptor orsoluble mouse Zcytor16 heterodimeric polypeptide, such as soluble mouseZcytor16/CRF2-4, can be prepared as a fusion to a dimerizing protein asdisclosed in U.S. Pat. Nos. 5,155,027 and 5,567,584. Preferreddimerizing proteins in this regard include immunoglobulin constantregion domains, e.g., IgGγl, and the human κ light chain. Immunoglobulinsoluble mouse Zcytor16 receptor or immunoglobulin-soluble mouse Zcytor16heterodimeric or multimeric polypeptide, such as immunoglobulin-solublemouse Zcytor16/CRF2-4 fusions can be expressed in genetically engineeredcells to produce a variety of multimeric mouse Zcytor16 receptoranalogs. Auxiliary domains can be fused to soluble mouse Zcytor16receptor or soluble mouse Zcytor16 heterodimeric or multimericpolypeptides, such as soluble mouse Zcytor16/CRF2-4 to target them tospecific cells, tissues, or macromolecules (e.g., collagen, or cellsexpressing the Zcytor16 ligand, IL-TIF). A mouse Zcytor16 polypeptidecan be fused to two or more moieties, such as an affinity tag forpurification and a targeting domain. Polypeptide fusions can alsocomprise one or more cleavage sites, particularly between domains. See,Tuan et al., Connective Tissue Research 34:1-9, 1996.

In bacterial cells, it is often desirable to express a heterologousprotein as a fusion protein to decrease toxicity, increase stability,and to enhance recovery of the expressed protein. For example, mouseZcytor16 can be expressed as a fusion protein comprising a glutathioneS-transferase polypeptide. Glutathione S-transferease fusion proteinsare typically soluble, and easily purifiable from E. coli lysates onimmobilized glutathione columns. In similar approaches, a mouse Zcytor16fusion protein comprising a maltose binding protein polypeptide can beisolated with an amylose resin column, while a fusion protein comprisingthe C-terminal end of a truncated Protein A gene can be purified usingIgG-Sepharose. Established techniques for expressing a heterologouspolypeptide as a fusion protein in a bacterial cell are described, forexample, by Williams et al., “Expression of Foreign Proteins in E. coliUsing Plasmid Vectors and Purification of Specific PolyclonalAntibodies,” in DNA Cloning 2: A Practical Approach, 2^(nd) Edition,Glover and Hames (Eds.), pages 15-58 (Oxford University Press 1995). Inaddition, commercially available expression systems are available. Forexample, the PINPOINT Xa protein purification system (PromegaCorporation; Madison, Wis.) provides a method for isolating a fusionprotein, comprising a polypeptide that becomes biotihylated duringexpression with a resin that comprises avidin.

Peptide tags that are useful for isolating heterologous polypeptidesexpressed by either prokaryotic or eukaryotic cells includepolymistidine tags (which have an affinity for nickel-chelating resin),c-myc tags, calmodulin binding protein (isolated with calmodulinaffinity chromatography), substance P, the RYIRS tag (which binds withanti-RYIRS antibodies), the Glu-Glu tag, and the FLAG tag (which bindswith anti-FLAG antibodies). See, for example, Luo et al., Arch. Biochem.Biophys. 329:215 (1996), Morganti et al., Biotechnol. Appl. Biochem.23:67 (1996), and Zheng et al., Gene 186:55 (1997). Nucleic acidmolecules encoding such peptide tags are available, for example, fromSigma-Aldrich Corporation (St. Louis, Mo.).

The present invention also contemplates that the use of the secretorysignal sequence contained in the mouse Zcytor16 polypeptides of thepresent invention to direct other polypeptides into the secretorypathway. A signal fusion polypeptide can be made wherein a secretorysignal sequence derived from amino acid residues 1 to 23 of SEQ ID NO:38or SEQ ID NO:48 is operably linked to another polypeptide using methodsknown in the art and disclosed herein. The secretory signal sequencecontained in the fusion polypeptides of the present invention ispreferably fused amino-terminally to an additional peptide to direct theadditional peptide into the secretory pathway. Such constructs havenumerous applications known in the art. For example, these novelsecretory signal sequence fusion constructs can direct the secretion ofan active component of a normally non-secreted protein, such as areceptor. Such fusions may be used in a transgenic animal or in acultured recombinant host to direct peptides through the secretorypathway. With regard to the latter, exemplary polypeptides includepharmaceutically active molecules such as Factor VIIa, proinsulin,insulin, follicle stimulating hormone, tissue type plasminogenactivator, tumor necrosis factor, interleukins (e.g., interleukin-1(IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, L-13, IL-14, and IL-15), colony stimulating factors (e.g.,granulocyte-colony stimulating factor (G-CSF) and granulocytemacrophage-colony stimulating factor (GM-CSF)), interferons (e.g.,interferons-α, -β, -γ, -ω, -δ and -τ), the stem cell growth factordesignated “S1 factor,” erythropoietin, and thrombopoietin. The mouseZcytor16 secretory signal sequence contained in the fusion polypeptidesof the presents invention is preferably fused amino-terminally to anadditional peptide to direct the additional peptide into the secretorypathway. Fusion proteins comprising a mouse Zcytor16 secretory signalsequence can be constructed using standard techniques.

Another form of fusion protein comprises a mouse Zcytor16 polypeptideand an immunoglobulin heavy chain constant region, typically an F_(c)fragment, which contains two or three constant region domains and ahinge region but lacks the variable region. As an illustration, Chang etal., U.S. Pat. No. 5,723,125, describe a fusion protein comprising ahuman interferon and a human immunoglobulin Fc fragment. The C-terminalof the interferon is linked to the N-terminal of the Fc fragment by apeptide linker moiety. An example of a peptide linker is a peptidecomprising primarily a T cell inert sequence, which is immunologicallyinert. An exemplary peptide linker has the amino acid sequence: GGSGGSGGGG SGGGG S (SEQ ID NO:4). In this fusion protein, an illustrative Fcmoiety is a human γ4 chain, which is stable in solution and has littleor no complement activating activity. Accordingly, the present inventioncontemplates a mouse Zcytor16 fusion protein that comprises a mouseZcytor16 moiety and a human Fc fragment, wherein the C-terminus of themouse Zcytor16 moiety is attached to the N-terminus of the Fc fragmentvia a peptide linker, such as a peptide consisting of the amino acidsequence of SEQ ID NO:4. The mouse Zcytor16 moiety can be a mouseZcytor16 molecule or a fragment thereof. For example, a fusion proteincan comprise amino acid residues 24 to 230, or 27 to 230 of SEQ ID NO:38or SEQ ID NO:48 and an Fc fragment (e.g., a human Fc fragment).

In another variation, a mouse Zcytor16 fusion protein comprises an IgGsequence, a mouse Zcytor16 moiety covalently joined to the aminoterminalend of the IgG sequence, and a signal peptide that is covalently joinedto the aminoterminal of the mouse Zcytor16 moiety, wherein the IgGsequence consists of the following elements in the following order: ahinge region, a CH₂ domain, and a CH₃ domain. Accordingly, the IgGsequence lacks a CH₁ domain. The mouse Zcytor16 moiety displays a mouseZcytor16 activity, as described herein, such as the ability to bind witha Zcytor16 ligand, including mouse and human IL-TIF. This generalapproach to producing fusion proteins that comprise both antibody andnonantibody portions has been described by LaRochelle et al., EP 742830(WO 95/21258).

Fusion proteins comprising a mouse Zcytor16 moiety and an Fc moiety canbe used, for example, as an in vitro assay tool. For example, thepresence of a Zcytor16 ligand, including mouse or human IL-TIF, in abiological sample can be detected using a mouse Zcytor16-immunoglobulinfusion protein, in which the mouse Zcytor16 moiety is used to bind theligand, and a macromolecule, such as Protein A or anti-Fc antibody, isused to bind the fusion protein to a solid support. Such systems can beused to identify agonists and antagonists that interfere with thebinding of a Zcytor16 ligand to its receptor.

Other examples of antibody fusion proteins include polypeptides thatcomprise an antigen-binding domain and a mouse Zcytor16 fragment thatcontains a mouse Zcytor16 extracellular domain. Such molecules can beused to target particular tissues for the benefit of Zcytor16 bindingactivity.

The present invention further provides a variety of other polypeptidefusions. For example, part or all of a domain(s) conferring a biologicalfunction can be swapped between mouse Zcytor16 of the present inventionwith the functionally equivalent domain(s) from another member of thecytokine receptor family. Polypeptide fusions can be expressed inrecombinant host cells to produce a variety of mouse Zcytor16 fusionanalogs. A mouse Zcytor16 polypeptide can be fused to two or moremoieties or domains, such as an affinity tag for purification and atargeting domain. Polypeptide fusions can also comprise one or morecleavage sites, particularly between domains. See, for example, Tuan etal., Connective Tissue Research 34:1 (1996).

Fusion proteins can be prepared by methods known to those skilled in theart by preparing each component of the fusion protein and chemicallyconjugating them. Alternatively, a polynucleotide encoding bothcomponents of the fusion protein in the proper reading frame can begenerated using known techniques and expressed by the methods describedherein. General methods for enzymatic and chemical cleavage of fusionproteins are described, for example, by Ausubel (1995) at pages 16-19 to16-25.

Mouse Zcytor16 polypeptides can be used to identify and to isolateZcytor16 ligands; including mouse and-human IL-TIF. For example,proteins and peptides of the present invention can be immobilized on acolumn and used to bind ligands from a biological sample that is runover the column (Hermanson et al. (eds.), Immobilized Affinity LigandTechniques, pages 195-202 (Academic Press 1992)). As such, mouseZcytor16 polypeptides of the present invention can be used to identifyand isolate IL-TIF for either diagnostic, or production purposes.

The activity of a mouse Zcytor16 polypeptide can also be observed by asilicon-based biosensor microphysiometer, which measures theextracellular acidification rate or proton excretion associated withreceptor binding and subsequent physiologic cellular responses. Anexemplary device is the CYTOSENSOR Microphysiometer manufactured byMolecular Devices, Sunnyvale, Calif. A variety of cellular responses,such as cell proliferation, ion transport, energy production,inflammatory response, regulatory and receptor activation, and the like,can be measured by this method (see, for example, McConnell et al.,Science 257:1906 (1992), Pitchford et al., Meth. Enzymol. 228:84 (1997),Arimilli et al., J. Immunol. Meth. 212:49 (1998), Van Liefde et al.,Eur. J. Pharmacol. 346:87 (1998)). The microphysiometer can be used forassaying eukaryotic, prokaryotic, adherent, or non-adherent cells. Bymeasuring extracellular acidification changes in cell media over time,the microphysiometer directly measures cellular responses to variousstimuli, including agonists, ligands, or antagonists of mouse Zcytor16.

For example, the microphysiometer is used to measure responses of anmouse Zcytor16-expressing eukaryotic cell, compared to a controleukaryotic cell that does not express mouse Zcytor16 polypeptide.Suitable cells responsive to mouse Zcytor16-modulating stimuli includerecombinant host cells comprising a mouse Zcytor16 expression vector,and cells that naturally express mouse Zcytor16. Extracellularacidification provides one measure for a mouse Zcytor16-modulatedcellular response. In addition, this approach can be used to identifyligands, agonists, and antagonists of Zcytor16 ligand, IL-TIF. Forexample, a molecule can be identified as an agonist of Zcytor16 ligandby providing cells that express a mouse Zcytor16 polypeptide, culturinga first portion of the cells in the absence of the test compound,culturing a second portion of the cells in the presence of the testcompound, and determining whether the second portion exhibits a cellularresponse, in comparison with the first portion.

Alternatively, a solid phase system can be used to identify a Zcytor16ligand, or an agonist or antagonist of a Zcytor16 ligand. For example, amouse Zcytor16 polypeptide or mouse Zcytor16 fusion protein, or mouseZcytor16 monomeric, homodimeric, heterodimeric or multimeric solublereceptor can be immobilized onto the surface of a receptor chip of acommercially available biosensor instrument (BIACORE, Biacore AB;Uppsala, Sweden). The use of this instrument is disclosed, for example,by Karlsson, Immunol. Methods 145:229 (1991), and Cunningham and Wells,J. Mol. Biol. 234:554 (1993).

In brief, a mouse Zcytor16 polypeptide or fusion protein is covalentlyattached, using amine or sulfhydryl chemistry, to dextran fibers thatare attached to gold film within a flow cell. A test sample is thenpassed through the cell. If a ligand is present in the sample, it willbind to the immobilized polypeptide or fusion protein, causing a changein the refractive index of the medium, which is detected as a change insurface plasmon resonance of the gold film. This system allows thedetermination of on- and off-rates, from which binding affinity can becalculated, and assessment of stoichiometry of binding. This system canalso be used to examine antibody-antigen interactions, and theinteractions of other complement/anti-complement pairs.

Mouse Zcytor16 binding domains can be further characterized by physicalanalysis of structure, as determined by such techniques as nuclearmagnetic resonance, crystallography, electron diffraction orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids of Zcytor16 ligand agonists. See, for example, de Voset al., Science 255:306 (1992), Smith et al., J. Mol. Biol. 224:899(1992), and Wlodaver et al., FEBS Lett. 309:59 (1992).

The present invention also contemplates chemically modified mouseZcytor16 compositions, in which a mouse Zcytor16 polypeptide is linkedwith a polymer. Illustrative mouse Zcytor16 polypeptides are solublepolypeptides that lack a functional transmembrane domain, such as apolypeptide consisting of amino acid residues 24 to 230, or 27 to 230 ofSEQ ID NO:38 or SEQ ID NO:48. Typically, the polymer is water soluble sothat the mouse Zcytor16 conjugate does not precipitated in an aqueousenvironment, such as a physiological environment. An example of asuitable polymer is one that has been modified to have a single reactivegroup, such as an active ester for acylation, or an aldehyde foralkylation, In this way, the degree of polymerization can be controlled.An example of a reactive aldehyde is polyethylene glycolpropionaldehyde, or mono-(C1-C10) alkoxy, or aryloxy derivatives thereof(see, for example, Harris, et al., U.S. Pat. No. 5,252,714). The polymermay be branched or unbranched. Moreover, a mixture of polymers can beused to produce mouse Zcytor16 conjugates.

Mouse Zcytor16 conjugates used for therapy can comprise pharmaceuticallyacceptable water-soluble polymer moieties. Suitable water-solublepolymers include polyethylene glycol (PEG), monomethoxy-PEG,mono-(C1-C10)alkoxy-PEG, aryloxy-PEG, poly-(N-vinyl pyrrolidone)PEG,tresyl monomethoxy PEG, PEG propionaldehyde, bis-succinimidyl carbonatePEG, propylene glycol homopolymers, a polypropylene oxide/ethylene oxideco-polymer, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, dextran, cellulose, or other carbohydrate-based polymers.Suitable PEG may have a molecular weight from about 600 to about 60,000,including, for example, 5,000, 12,000, 20,000 and 25,000. A mouseZcytor16 conjugate can also comprise a mixture of such water-solublepolymers.

One example of a mouse Zcytor16 conjugate comprises a mouse Zcytor16moiety and a polyalkyl oxide moiety attached to the N-terminus of themouse Zcytor16 moiety. PEG is one suitable polyalkyl oxide. As anillustration, mouse Zcytor16 can be modified with PEG, a process knownas “PEGylation.” PEGylation of mouse Zcytor16 can be carried out by anyof the PEGylation reactions known in the art (see, for example, EP 0 154316, Delgado et al., Critical Reviews in Therapeutic Drug CarrierSystems 9:249 (1992), Duncan and Spreafico, Clin. Pharmacokinet. 27:290(1994), and Francis et al., Int J Hematol 68:1 (1998)). For example,PEGylation can be performed by an acylation reaction or by an alkylationreaction with a reactive polyethylene glycol molecule. In an alternativeapproach, mouse Zcytor16 conjugates are formed by condensing activatedPEG, in which a terminal hydroxy or amino group of PEG has been replacedby an activated linker (see, for example, Karasiewicz et al., U.S. Pat.No. 5,382,657).

PEGylation by acylation typically requires reacting an active esterderivative of PEG with a mouse Zcytor16 polypeptide. An example of anactivated PEG ester is PEG esterified to N-hydroxysuccinimide. As usedherein, the term “acylation” includes the following types of linkagesbetween mouse Zcytor16 and a water soluble polymer: amide, carbamate,urethane, and the like. Methods for preparing PEGylated mouse Zcytor16by acylation will typically comprise the steps of (a) reacting a mouseZcytor16 polypeptide with PEG (such as a reactive ester of an aldehydederivative of PEG) under conditions whereby one or more PEG groupsattach to mouse Zcytor16, and (b) obtaining the reaction product(s).Generally, the optimal reaction conditions for acylation reactions willbe determined based upon known parameters and desired results. Forexample, the larger the ratio of PEG:mouse Zcytor16, the greater thepercentage of polyPEGylated mouse Zcytor16 product.

The product of PEGylation by acylation is typically a polyPEGylatedmouse Zcytor16 product, wherein the lysine ε-amino groups are PEGylatedvia an acyl linking group. An example of a connecting linkage is anamide. Typically, the resulting mouse Zcytor16 will be at least 95%mono-, di-, or tri-pegylated, although some species with higher degreesof PEGylation may be formed depending upon the reaction conditions.PEGylated species can be separated from unconjugated mouse Zcytor16polypeptides using standard purification methods, such as dialysis,ultrafiltration, ion exchange chromatography, affinity chromatography,and the like.

PEGylation by alkylation generally involves reacting a terminal aldehydederivative of PEG with mouse Zcytor16 in the presence of a reducingagent. PEG groups can be attached to the polypeptide via a —CH₂—NHgroup.

Derivatization via reductive alkylation to produce a monoPEGylatedproduct takes advantage of the differential reactivity of differenttypes of primary amino groups available for derivatization. Typically,the reaction is performed at a pH that allows one to take advantage ofthe pKa differences between the ε-amino groups of the lysine residuesand the α-amino group of the N-terminal residue of the protein. By such,selective derivatization, attachment of a water-soluble polymer thatcontains a reactive group such as an aldehyde, to a protein iscontrolled. The conjugation with the polymer occurs predominantly at theN-terminus of the protein without significant modification of otherreactive groups such as the lysine side chain amino groups. The presentinvention provides a substantially homogenous preparation of mouseZcytol16 monopolymer conjugates.

Reductive alkylation to produce a substantially homogenous population ofmonopolymer mouse Zcytor16 conjugate molecule can comprise the steps of:(a) reacting a mouse Zcytor16 polypeptide with a reactive PEG underreductive alkylation conditions at a pH suitable to permit selectivemodification of the α-amnino group at the amino terminus of the mouseZcytor16, and (b) obtaining the reaction product(s). The reducing agentused for reductive alkylation should be stable in aqueous solution andable to reduce only the Schiff base formed in the initial process ofreductive alkylation. Illustrative reducing agents include sodiumborohydride, sodium cyanoborohydride, dimethylamine borane,trimethylamine borane, and pyridine borane.

For a substantially homogenous population of monopolymer mouse Zcytor16conjugates, the reductive alkylation reaction conditions are those thatpermit the selective attachment of the water-soluble polymer moiety tothe N-terminus of mouse Zcytor16. Such reaction conditions generallyprovide for pKa differences between the lysine amino groups and theα-amino group at the N-terminus. The pH also affects the ratio ofpolymer to protein to be used. In general, if the pH is lower, a largerexcess of polymer to protein will be desired because the less reactivethe N-terminal α-group, the more polymer is needed to achieve optimalconditions. If the pH is higher, the polymer:mouse Zcytor16 need not beas large because more reactive groups are available. Typically, the pHwill fall within the range of 3 to 9, or 3 to 6. This method can beemployed for making mouse Zcytor16-comprising homodimeric, heterodimericor multimeric soluble receptor conjugates.

Another factor to consider is the molecular weight of the water-solublepolymer. Generally, the higher the molecular weight of the polymer, thefewer number of polymer molecules which may be attached to the protein.For PEGylation reactions, the typical molecular weight is about 2 kDa toabout 100 kDa, about 5 kDa to about 50 kDa, or about 12 kDa to about 25kDa. The molar ratio of water-soluble polymer to mouse Zcytor16 willgenerally be in the range of 1:1 to 100:1. Typically, the molar ratio ofwater-soluble polymer to mouse Zcytor16 will be 1:1 to 20:1 forpolyPEGylation, and 1:1 to5:1for monoPEGylation.

General methods for producing conjugates comprising a polypeptide andwater-soluble polymer moieties are known in the art. See, for example,Karasiewicz et al., U.S. Pat. No. 5,382,657, Greenwald et al., U.S. Pat.No. 5,738, 846, Nieforth et al., Clin. Pharmacol. Ther. 59:636 (1996),Monkarsh et al., Anal. Biochem. 247:434 (1997)). This method can beemployed for making mouse Zcytor16-comprising homodimeric, heterodimericor multimeric soluble receptor conjugates.

The present invention contemplates compositions comprising a peptide orpolypeptide described herein. Such compositions can further comprise acarrier. The carrier can be a conventional organic or inorganic carrier.Examples of carriers include water, buffer solution, alcohol, propyleneglycol, macrogol, sesame oil, corn oil, and the like.

8. Isolation of Mouse Zcytor16 Polypeptides

The polypeptides of the present invention can be purified to at leastabout 80% purity, to at least about 90% purity, to at least about 95%purity, or greater than 95% purity with respect to contaminatingmacromolecules, particularly other proteins and nucleic acids, and freeof infectious and pyrogenic agents. The polypeptides of the presentinvention may also be purified to a pharmaceutically pure state, whichis greater than 99.9% pure. In certain preparations, purifiedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin.

Fractionation and/or conventional purification methods can be used toobtain preparations of mouse Zcytor16 purified from natural sources(e.g., tonsil tissue), synthetic mouse Zcytor16 polypeptides, andrecombinant mouse Zcytor16 polypeptides and fusion mouse Zcytor16polypeptides purified from recombinant host cells. In general, ammoniumsulfate precipitation and acid or chaotrope extraction may be used forfractionation of samples. Exemplary purification steps may includehydroxyapatite, size exclusion, FPLC and reverse-phase high performanceliquid chromatography. Suitable chromatographic media includederivatized dextrans, agarose, cellulose, polyacrylamide, specialtysilicas, and the like. PEI, DEAE, QAE and Q derivatives are suitable.Exemplary chromatographic media include those media derivatized withphenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia),Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose(Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG71 (Toso Haas) and the like. Suitable solid supports include glassbeads, silica-based resins, cellulosic resins, agarose beads,cross-linked agarose beads, polystyrene beads, cross-linkedpolyacrylamide resins and the like that are insoluble under theconditions in which they are to be used. These supports may be modifiedwith reactive groups that allow attachment of proteins by amino groups,carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydratemoieties.

Examples of coupling chemistries include cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, hydrazide activation, and carboxyl and amino derivatives forcarbodiimide coupling chemistries. These and other solid media are wellknown and widely used in the art, and are available from commercialsuppliers. Selection of a particular method for polypeptide isolationand purification is a matter of routine design and is determined in partby the properties of the chosen support. See, for example, AffinityChromatography: Principles & Methods (Pharmacia LKB Biotechnology 1988),and Doonan, Protein Purification Protocols (The Humana Press 1996).

Additional variations in mouse Zcytor16 isolation and purification canbe devised by those of skill in the art. For example, anti-mouseZcytor16 antibodies, obtained as described below, can be used to isolatelarge quantities of protein by immunoaffinity purification.

The polypeptides of the present invention can also be isolated byexploitation of particular properties. For example, immobilized metalion adsorption (IMAC) chromatography can be used to purifyhistidine-rich proteins, including those comprising polyhistidine tags.Briefly, a gel is first charged with divalent metal ions to form achelate (Sulkowski, Trends in Biochem. 3:1 (1985)). Histidine-richproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include, purification of glycosylated proteinsby lectin affinity chromatography and ion exchange chromatography (M.Deutscher, (ed.), Meth. Enzymol. 182:529 (1990)). Within additionalembodiments of the invention, a fusion of the polypeptide of interestand an affinity tag (e.g., maltose-binding protein, an immunoglobulindomain) may be constructed to facilitate purification. Moreover, theligand-binding properties of mouse Zcytor16 extracellular domain can beexploited for purification, for example, of mouse Zcytor16-comprisingsoluble receptors; for example, by using affinity chromatography whereinIL-TIF ligand is bound to a column and the mouse Zcytor16-comprisingreceptor is bound and subsequently eluted using standard chromatographymethods.

mouse Zcytor16 polypeptides or fragments thereof may also be preparedthrough chemical synthesis, as described above. mouse Zcytor16polypeptides may be monomers or multimers; glycosylated ornon-glycosylated; PEGylated or non-PEGylated; and may or may not includean initial methionine amino acid residue.

9. Production of Antibodies to Mouse Zcytor16 Proteins

Antibodies to mouse Zcytor16 can be obtained, for example, using theproduct of a mouse Zcytor16 expression vector or mouse Zcytor16 isolatedfrom a natural source as an antigen. Particularly useful anti-mouseZcytor16 antibodies “bind specifically” with mouse Zcytor16. Antibodiesare considered to be specifically binding if the antibodies exhibit atleast one of the following two properties: (1) antibodies bind to mouseZcytor16 with a threshold level of binding activity, and (2) antibodiesdo not significantly cross-react with polypeptides related to mouseZcytor16.

With regard to the first characteristic, antibodies specifically bind ifthey bind to a mouse Zcytor16 polypeptide, peptide or epitope with abinding affinity (K_(a)) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ orgreater, more preferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹or greater. The binding affinity of an antibody can be readilydetermined by one of ordinary skill in the art, for example, byScatchard analysis (Scatchard, Ann. NY Acad. Sci. 51:660 (1949)). Withregard to the second characteristic, antibodies do not significantlycross-react with related polypeptide molecules, for example, if theydetect mouse Zcytor16, but not presently known polypeptides using astandard Western blot analysis. Examples of known related polypeptidesinclude known cytokine receptors.

Anti-mouse Zcytor16 antibodies can be produced using antigenic mouseZcytor16 epitope-bearing peptides and polypeptides. Antigenicepitope-bearing peptides and polypeptides of the present inventioncontain a sequence of at least nine, or between 15 to about 30 aminoacids contained within SEQ ID NO:38 or SEQ ID NO:48 or another aminoacid sequence disclosed herein. However, peptides or polypeptidescomprising a larger portion of an amino acid sequence of the invention,containing from 30 to 50 amino acids, or any length up to and includingthe entire amino acid sequence of a polypeptide of the invention, alsoare useful for inducing antibodies that bind with mouse Zcytor16. It isdesirable that the amino acid sequence of the epitope-bearing peptide isselected to provide substantial solubility in aqueous solvents (i.e.,the sequence includes relatively hydrophilic residues, while hydrophobicresidues are typically avoided). Moreover, amino acid sequencescontaining proline residues may be also be desirable for antibodyproduction.

As an illustration, potential antigenic sites in mouse Zcytor16 wereidentified using the Jameson-Wolf method, Jameson and Wolf, CABIOS4:181, (1988), as implemented by the PROTEAN program (version 3.14) ofLASERGENE (DNASTAR; Madison, Wis.). Default parameters were used in thisanalysis.

The Jameson-Wolf method predicts potential antigenic determinants bycombining six major subroutines for protein structural prediction.Briefly, the Hopp-Woods method, Hopp et al., Proc. Nat'l Acad. Sci. USA78:3824 (1981), was first used to identify amino acid sequencesrepresenting areas of greatest local hydrophilicity (parameter: sevenresidues averaged). In the second step, Emini's method, Emini et al., J.Virology 55:836 (1985), was used to calculate surface probabilities(parameter: surface decision threshold (0.6)=1). Third, theKarplus-Schultz method, Karplus and Schultz, Naturwissenschaften 72:212(1985), was used to predict backbone chain flexibility (parameter:flexibility threshold (0.2)=1). In the fourth and fifth steps of theanalysis, secondary structure predictions were applied to the data usingthe methods of Chou-Fasman, Chou, “Prediction of Protein StructuralClasses from Amino Acid Composition,” in Prediction of Protein Structureand the Principles of Protein Conformation, Fasman (ed.), pages549-586.(Plenum Press 1990), and Garnier-Robson, Gamier et al., J. Mol.Biol. 120:97 (1978) (Chou-Fasman parameters: conformation table=64proteins; α region threshold=103; β region threshold=105; Gamier-Robsonparameters: α and β decision constants=0). In the sixth subroutine,flexibility parameters and hydropathy/solvent accessibility factors werecombined to determine a surface contour value, designated as the“antigenic index.” Finally, a peak broadening function was applied tothe antigenic index, which broadens major surface peaks by adding 20,40, 60, or 80% of the respective peak value to account for additionalfree energy derived from the mobility of surface regions relative tointerior regions. This calculation was not applied, however, to anymajor peak that resides in a helical region, since helical regions tendto be less flexible.

The results of this analysis indicated that the following amino acidsequences of SEQ ID NO:38 or SEQ ID NO:48 would provide suitableantigenic peptides: amino acids 35 to 40 (“antigenic peptide 1”), aminoacids 67 to 77 (“antigenic peptide 2”), 88 to 94 (“antigenic peptide3”), amino acids 108 to 119 (“antigenic peptide 4”), amino acids 108 to130 (“antigenic peptide 5”), amino acids 125 to 130 (“antigenic peptide6”), amino acids 147 to 161 (“antigenic peptide 7”), amino acids 177 to190 (“antigenic peptide 8”), and amino acids 216 to 225 (“antigenicpeptide 9”). The present invention contemplates the use of any one ofantigenic peptides 1 to 9 to generate antibodies to mouse Zcytor16. Thepresent invention also contemplates polypeptides comprising at least oneof antigenic peptides 1 to 9. A Hopp/Woods hydrophilicity profile of theZcytor16 protein sequence as shown in SEQ ID NO:38 or SEQ ID NO:48 canbe generated (Hopp et al., Proc. Natl. Acad. Sci.78:3824-3828, 1981;Hopp, J. Immun. Meth. 88:1-18, 1986 and Triquier et al., ProteinEngineering 11:153-169, 1998). The profile is based on a slidingsix-residue window. Buried G, S, and T residues and exposed H, Y, and Wresidues were ignored. For example, in Zcytor16, hydrophilic regionsthat serve as suitable antigens, include in reference to SEQ ID NO:38 orSEQ ID NO:48: (1) amino acid number 220 to 225; (2) amino acid number216 to 221; (3) amino acid number 180 to 185; (4) amino acid number 179to 184; and (5) amino acid number 71 to 76. Moreover other suitableantigens comprise residues, 24, 27 or 31 to 122 or 126, 131to229 or 230;1 to 229 or 230; and 24 to to 229 or 230 of SEQ ID NO:38 or SEQ IDNO:48.

Moreover, suitable antigens also include the mouse Zcytor16 polypeptidescomprising a mouse Zcytor16 cytokine binding, or extracellular domaindisclosed above in combination with another class I or II cytokineextracellular domain, such as those that form soluble mouse Zcytor16heterodimeric or multimeric polypeptides, such as soluble mouseZcytor16/CRF2-4, mouse Zcytor16/zcytor11, mouse Zcytor16/zcytor7, andthe like.

Polyclonal antibodies to recombinant mouse Zcytor16 protein or to mouseZcytor16 isolated from natural sources can be prepared using methodswell-known to those of skill in the art. See, for example, Green et al.,“Production of Polyclonal Antisera,” in Immunochemical Protocols(Manson, ed.), pages 1-5 (Humana Press 1992), and Williams et al.,“Expression of foreign proteins in E. coli using plasmid vectors andpurification of specific polyclonal antibodies,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (OxfordUniversity Press 1995). The immunogenicity of a mouse Zcytor16polypeptide can be increased through the use of an adjuvant, such asalum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.Polypeptides useful for immunization also include fusion polypeptides,such as fusions of mouse Zcytor16 or a portion thereof with animmunoglobulin polypeptide or with maltose binding protein. Thepolypeptide immunogen may be a full-length molecule or a portionthereof. If the polypeptide portion is “hapten-like,” such portion maybe advantageously joined or linked to a macromolecular carrier (such askeyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanustoxoid) for immunization.

Although polyclonal antibodies are typically raised in animals such ashorses, cows, dogs, chicken, rats, mice, rabbits, guinea pigs, goats, orsheep, an anti-mouse Zcytor16 antibody of the present invention may alsobe derived from a subhuman primate antibody. General techniques forraising diagnostically and therapeutically useful antibodies in baboonsmay be found, for example, in Goldenberg et al., international patentpublication, No. WO 91/11465, and in Losman et al., Int. J. Cancer46:310 (1990).

Alternatively, monoclonal anti-mouse Zcytor16 antibodies can begenerated. Rodent monoclonal antibodies to specific antigens may beobtained by methods known to those skilled in the art (see, for example,Kohler et al., Nature 256:495 (1975), Coligan et al. (eds.), CurrentProtocols in Immunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991) [“Coligan”], Picksley et al., “Production of monoclonal antibodiesagainst proteins expressed in E. coli,” in DNA Cloning 2: ExpressionSystems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford UniversityPress 1995)).

Briefly, monoclonal antibodies can be obtained by injecting mice with acomposition comprising a mouse Zcytor16 gene product, verifying thepresence of antibody production by removing a serum sample, removing thespleen to obtain B-lymphocytes, fusing the B-lymphocytes with myelomacells to produce hybridomas, cloning the hybridomas, selecting positiveclones which produce antibodies to the antigen, culturing the clonesthat produce antibodies to the antigen, and isolating the antibodiesfrom the hybridoma cultures.

In addition, an anti-mouse Zcytor16 antibody of the present inventionmay be derived from a human monoclonal antibody. Human monoclonalantibodies are obtained from transgenic mice that have been engineeredto produce specific human antibodies in response to antigenic challenge.In this technique, elements of the human heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous heavy chain andlight chain loci. The transgenic mice can synthesize human antibodiesagainst specific antigens, e.g., Zcytor16, and the mice can be used toproduce human antibody-secreting hybridomas. Methods for obtaining humanantibodies from transgenic mice are described, for example, by Green etal., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994),and Taylor et al., Int. Immun. 6:579 (1994).

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography (see, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines etal., “Purification of Immunoglobulin G (IgG),” in Methods in MolecularBiology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)).

For particular uses, it may be desirable to prepare fragments ofanti-mouse Zcytor16 antibodies. Such antibody fragments can be obtained,for example, by proteolytic hydrolysis of the antibody. Antibodyfragments can be obtained by pepsin or papain digestion of wholeantibodies by conventional methods. As an illustration, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent to produce 3.5S Fab′monovalent fragments. Optionally, the cleavage reaction can be performedusing a blocking group for the sulfhydryl groups that result fromcleavage of disulfide linkages. As an alternative, an enzymatic cleavageusing pepsin produces two monovalent Fab fragments and an Fc fragmentdirectly. These methods are described, for example, by Goldenberg, U.S.Pat. No. 4,331,647, Nisonoff et al., Arch Biochem. Biophys. 89:230(1960), Porter, Biochem. J. 73:119 (1959), Edelman et al., in Methods inEnzymology Vol. 1, page 422 (Academic Press 1967), and by Coligan atpages 2.8.1-2.8.10 and 2.10.-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association can be noncovalent, as described by Inbar etal., Proc. Nat'l Acad. Sci. USA 69:2659 (1972). Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde (see, for example,Sandhu, Crit. Rev. Biotech. 12:437 (1992)).

The Fv fragments may comprise V_(H) and V_(L) chains which are connectedby a peptide linker. These single-chain antigen binding proteins (scFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains which are connected by anoligonucleotide. The structural gene is inserted into an expressionvector which is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingscFvs are described, for example, by Whitlow et al., Methods: ACompanion to Methods in Enzymology 2:97 (1991) (also see, Bird et al.,Science 242:423 (1988), Ladner et al., U.S. Pat. No. 4,946,778, Pack etal., Bio/Technology 11:1271 (1993), and Sandhu, supra).

As an illustration, a scFV can be obtained by exposing lymphocytes tomouse Zcytor16 polypeptide in vitro, and selecting antibody displaylibraries in phage or similar vectors (for instance, through use ofimmobilized or labeled mouse Zcytor16 protein or peptide). Genesencoding polypeptides having potential mouse Zcytor16 polypeptidebinding domains can be obtained by screening random peptide librariesdisplayed on phage (phage display) or on bacteria, such as E. coli.Nucleotide sequences encoding the polypeptides can be obtained in anumber of ways, such as through random mutagenesis and randompolynucleotide synthesis. These random peptide display libraries can beused to screen for peptides which interact with a known target which canbe a protein or polypeptide, such as a ligand or receptor, a biologicalor synthetic macromolecule, or organic or inorganic substances.Techniques for creating and screening such random peptide displaylibraries are known in the art (Ladner et al., U.S. Pat. No. 5,223,409,Ladner et al., U.S. Pat. No. 4,946,778, Ladner et al., U.S. Pat. No.5,403,484, Ladner et al., U.S. Pat. No. 5,571,698, and Kay et al., PhageDisplay of Peptides and Proteins (Academic Press, Inc. 1996)) and randompeptide display libraries and kits for screening such libraries areavailable commercially, for instance from CLONTECH Laboratories, Inc.(Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New EnglandBiolabs, Inc. (Beverly, Mass.), and Pharmacia LKB Biotechnology Inc.(Piscataway, N.J.). Random peptide display libraries can be screenedusing the mouse Zcytor16 sequences disclosed herein to identify proteinswhich bind to mouse Zcytor16.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology 2:106 (1991),Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” inMonoclonal Antibodies: Production; Engineering and Clinical Application,Ritter et al. (eds.), page 166 (Cambridge University Press 1995), andWard et al., “Genetic Manipulation and Expression of Antibodies,” inMonoclonal Antibodies: Principles and Applications, Birch et al.,(eds.), page 137 (Wiley-Liss, Inc. 1995)).

Alternatively, an anti-mouse Zcytor16 antibody may be derived from a“humanized” monoclonal antibody. Humanized monoclonal antibodies areproduced by transferring mouse complementary determining regions fromheavy and light variable chains of the mouse immunoglobulin into a humanvariable domain. Typical residues of human antibodies are thensubstituted in the framework regions of the murine counterparts. The useof antibody components derived from humanized monoclonal antibodiesobviates potential problems associated with the immunogenicity of murineconstant regions. General techniques for cloning murine immunoglobulinvariable domains are described, for example, by Orlandi et al., Proc.Nat'l Acad. Sci. USA 86:3833 (1989). Techniques for producing humanizedmonoclonal antibodies are described, for example, by Jones et al.,Nature 321:522 (1986), Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285(1992), Sandhu, Crit. Rev. Biotech. 12:437 (1992), Singer et al., J.Immun. 150:2844 (1993), Sudhir (ed.), Antibody Engineering Protocols(Humana Press, Inc. 1995), Kelley, “Engineering Therapeutic Antibodies,”in Protein Engineering: Principles and Practice, Cleland et al. (eds.),pages 399-434 (John Wiley & Sons, Inc. 1996), and by Queen et al., U.S.Pat. No. 5,693,762 (1997).

Polyclonal anti-idiotype antibodies can be prepared by immunizinganimals with anti-mouse Zcytor16 antibodies or antibody fragments, usingstandard techniques. See, for example, Green et al., “Production ofPolyclonal Antisera,” in Methods In Molecular Biology: ImmunochemicalProtocols, Manson (ed.), pages 1-12 (Humana Press 1992). Also, seeColigan at pages 2.4.1-2.4.7. Alternatively, monoclonal anti-idiotypeantibodies can be prepared using anti-mouse Zcytor16 antibodies orantibody fragments as immunogens with the techniques, described above.As another alternative, humanized anti-idiotype antibodies or subhumanprimate anti-idiotype antibodies can be prepared using theabove-described techniques. Methods for producing anti-idiotypeantibodies are described, for example, by Irie, U.S. Pat. No.5,208,146,.Greene, et. al., U.S. Pat. No. 5,637,1677, and Varthakavi andMinocha, J. Gen. Virol. 77:1875 (1996).

10. Use of Mouse Zcytor16 Nucleotide Sequences to Detect Gene Expressionand Gene Structure

Nucleic acid molecules can be used to detect the expression of a mouseZcytor16 gene in a biological sample. Suitable probe molecules includedouble-stranded nucleic acid molecules comprising the nucleotidesequence of SEQ ID NO:37 or SEQ ID NO:47, or a portion thereof, as wellas single-stranded nucleic acid molecules having the complement of thenucleotide sequence of SEQ ID NO:37 or SEQ ID NO:47, or a portionthereof. Probe molecules may be DNA, RNA, oligonucleotides, and thelike. As used herein, the term “portion” refers to at least eightnucleotides to at least 20 or more nucleotides. Illustrative probes bindwith regions of the mouse Zcytor16 gene that have a low sequencesimilarity to comparable regions in other cytokine receptor genes.

In a basic assay, a single-stranded probe molecule is incubated withRNA, isolated from a biological sample, under conditions of temperatureand ionic strength that promote base pairing between the probe andtarget mouse Zcytor16 RNA species. After separating unbound probe fromhybridized molecules, the amount of hybrids is detected.

In addition, as Zcytor16 expression in humans and mice istissue-specific, polynucleotide probes, anti-mouse Zcytor16 antibodies,and detection the presence of Zcytor16 polypeptides in tissue can beused to assess whether a specific tissue is present, for example, aftersurgery involving the excision of a diseased or cancerous tissue. Assuch, the polynucleotides, polypeptides, and antibodies of the presentinvention can be used as an aid to determine whether all spleen tissueis excised after surgery, for example, after surgery for spleen cancer.In such instances, it is especially important to remove all potentiallydiseased tissue to maximize recovery from the cancer, and to minimizerecurrence. Preferred embodiments include fluorescent, radiolabeled, orcalorimetrically labeled antibodies, that can be used in situ.

Moreover, anti-mouse. Zcytor16 antibodies and binding frangments can beused for tagging and sorting cells that specifically-express mouseZcytor16, such as mononuclear cells, lymphoid cells, e.g, activated CD4+T-cells and CD19+ B-cells, and other described herein. Such methods ofcell tagging and sorting are well known in the art (see, e.g.,“Molecular Biology of the Cell”, 3^(rd) Ed., Albert, B. et al. (GarlandPublishing, London & New York, 1994). One of skill in the art wouldrecognize the importance of separating cell tissue types to study cells,and the use of antibodies to separate specific cell tissue types.Basically, antibodies that bind to the surface of a cell type arecoupled to various matrices such as collagen, polysaccharide beads, orplastic to form an affinity surface to which only cells recognized bythe antibodies will adhere. The bound cells are then recovered byconventional techniques. Other methods involve separating cells by afluorescence-activated cell sorter (FACS). In this technique one labelscells with antibodies that are coupled to a fluorescent dye. The labeledcells are then separated from unlabeled cells in a FACS machine. In FACSsorting individual cells traveling in single file pass through a laserbeam and the fluorescence of each cell is measured. Slightly furtherdown-stream, tiny droplets, most containing either one or no cells, areformed by a vibrating nozzle. The droplets containing a single cell areautomatically give a positive or negative charge at the moment offormation, depending on whether the cell they contain is fluorescent,and then deflected by a strong electric field into an appropriatecontainer. Such machines can select 1 cell in 1000 and sort about 5000cells each second. This produces a uniform population of cells for cellculture.

One of skill in the art would recognize that the antibodies to the mouseZcytor16 polypeptides of the present invention are useful, because notall tissue types express the mouse Zcytor16 receptor and because it isimportant that biologists be able to separate specific cell types forfurther study and/or therapeutic re-implantation into the body. This isparticularly relevant in cells such as immune cells, wherein mouseZcytor16 is expressed.

Moreover, use of mouse Zcytor16 polynucleotide probes, anti-mouseZcytor16 antibodies, and detection the presence of mouse Zcytor16polypeptides in tissue can be used in the diagnosis and/or prevention ofspontaneous abortions, or to monitor placental health and function.Since mouse Zcytor16 is expressed in the placenta, it could play a rolein the critical functions of placenta, such as proliferation or survivalof trophoblast cells, and the like. Thus, mouse Zcytor16 could beessential for the function of the placenta, thus maturation of embryos.Therefore, a supplement of mouse Zcytor16 polypeptide, or anti-mouseZcytor16 antibodies may be beneficial in the prevention and treatment ofcertain types of spontaneous abortions, or premature birth of babiescaused by abnormal expression of Zcytor16 in the placenta, or as adiagnostic to assess the function of the placenta. For example, as mouseZcytor16 is normally expressed in placenta, the absence of mouseZcytor16 expression may be indicative of abnormal placenta function.Similarly, use of mouse Zcytor16 polynucleotide probes, anti-mouseZcytor16 antibodies, can be used in animal husbandry, for example incommercial mouse breeding colonies and transgenic mouse embryorecipients in the prevention and treatment of certain types ofspontaneous abortions, or premature birth of pups caused by abnormalexpression of mouse Zcytor16 in the placenta, or as a diagnostic toassess the function of the placenta.

Well-established hybridization methods of RNA detection include northernanalysis and dot/slot blot hybridization (see, for example, Ausubel(1995) at pages 4-1 to 4-27, and Wu et al. (eds.), “Analysis of GeneExpression at the RNA Level,” in Methods in Gene Biotechnology, pages225-239 (CRC Press, Inc. 1997)). Nucleic acid probes can be detectablylabeled with radioisotopes such as ³²P or ³⁵S. Alternatively, mouseZcytor16 RNA can be detected with a nonradioactive hybridization method(see, for example, Isaac (ed.), Protocols for Nucleic Acid Analysis byNonradioactive Probes (Humana Press, Inc. 1993)). Typically,nonradioactive detection is achieved by enzymatic conversion ofchromogenic or chemiluminescent substrates. Illustrative nonradioactivemoieties include biotin, fluorescein, and digoxigenin.

Mouse Zcytor16 oligonucleotide probes are also useful for in vivodiagnosis. As an illustration, ¹⁸F-labeled oligonucleotides can beadministered to a subject and visualized by positron emission tomography(Tavitian et al., Nature Medicine 4:467 (1998)).

Numerous diagnostic procedures take advantage of the polymerase chainreaction (PCR) to increase sensitivity of detection methods. Standardtechniques for performing PCR are well-known (see, generally, Mathew(ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991),White (ed.), PCR Protocols: Current Methods and Applications (HumanaPress, Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (HumanaPress, Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols(Humana Press, Inc. 1998), Lo (ed.), Clinical Applications of PCR(Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis (HumanaPress, Inc. 1998)).

PCR primers can be designed to amplify a portion of the mouse Zcytor16gene that has a low sequence similarity to a comparable region in otherproteins, such as other cytokine receptor proteins.

One variation of PCR for diagnostic assays is reverse transcriptase-PCR(RT-PCR). In the RT-PCR technique, RNA is isolated from a biologicalsample, reverse transcribed to cDNA, and the cDNA is incubated withmouse Zcytor16 primers (see, for example, Wu et al. (eds.), “RapidIsolation of Specific cDNAs or Genes by PCR,” in Methods in GeneBiotechnology, pages 15-28 (CRC Press, Inc. 1997)). PCR is thenperformed and the products are analyzed using standard techniques.

As an illustration, RNA is isolated from biological sample using, forexample, the gunadinium-thiocyanate cell lysis procedure describedabove. Alternatively, a solid-phase technique can be used to isolatemRNA from a cell lysate. A reverse transcription reaction can be primedwith the isolated RNA using random oligonucleotides, short homopolymersof dT, or mouse Zcytor16 anti-sense oligomers. Oligo-dT primers offerthe advantage that various mRNA nucleotide sequences are amplified thatcan provide control target sequences. mouse Zcytor16 sequences areamplified by the polymerase chain reaction using two flankingoligonucleotide primers that are typically 20 bases in length.

PCR amplification products can be detected using a variety ofapproaches. For example, PCR products can be fractionated by gelelectrophoresis, and visualized by ethidium bromide staining.Alternatively, fractionated PCR products can be transferred to amembrane, hybridized with a detectably-labeled mouse Zcytor16 probe andexamined by autoradiography. Additional alternative approaches includethe use of digoxigenin-labeled deoxyribonucleic acid triphosphates toprovide chemiluminescence detection, and the C-TRAK colorimetric assay.

Another approach for detection of mouse Zcytor16 expression is cyclingprobe technology (CPT), in which a single-stranded DNA target binds withan excess of DNA-RNA-DNA chimeric probe to form a complex, the RNAportion is cleaved with RNAase H, and the presence of cleaved chimericprobe is detected (see, for example, Beggs et al., J. Clin. Microbiol.34:2985 (1996), Bekkaoui et al., Biotechniques 20:240 (1996)).Alternative methods for detection of mouse Zcytor16 sequences canutilize approaches such as nucleic acid sequence-based amplification(NASBA), cooperative amplification of templates by cross-hybridization(CATCH), and the ligase chain reaction (LCR) (see, for example, Marshallet al., U.S. Pat. No. 5,686,272 (1997), Dyer et al., J. Virol. Methods60:161 (1996), Ehricht et al., Eur. J. Biochem. 243:358 (1997), andChadwick et al., J. Virol. Methods 70:59 (1998)). Other standard methodsare known to those of skill in the art.

Mouse Zcytor16 probes and primers can also be used to detect and tolocalize Zcytor16 gene expression in tissue samples. Methods for such insitu hybridization are well-known to those of skill in the art (see, forexample, Choo (ed.), In Situ Hybridization Protocols (Humana Press, Inc.1994), Wu et al. (eds.), “Analysis of Cellular DNA or Abundance of mRNAby Radioactive In Situ Hybridization (RISH),” in Methods in GeneBiotechnology, pages 259-278 (CRC Press, Inc. 1997), and Wu et al.(eds.), “Localization of DNA or Abundance of mRNA by Fluorescence InSitu Hybridization (RISH),” in Methods in Gene Biotechnology, pages279-289 (CRC Press, Inc. 1997)). Various additional diagnosticapproaches are well-known to those of skill in the art (see, forexample, Mathew (ed.), Protocols in Human Molecular Genetics (HumanaPress, Inc. 1991), Coleman and Tsongalis, Molecular Diagnostics (HumanaPress, Inc. 1996), and Elles, Molecular Diagnosis of Genetic Diseases(Humana Press, Inc., 1996)). Suitable test samples include blood, urine,saliva, tissue biopsy, and autopsy material.

11. Use of Anti-Mouse Zcytor16 Antibodies to Detect Zcytor16 orAntagonize Zcytor16 Binding to IL-TIF

The present invention contemplates the use of anti-mouse Zcytor16antibodies to screen biological samples in vitro for the presence ofZcytor16. In one type of in vitro assay, anti-mouse Zcytor16 antibodiesare used in liquid phase. For example, the presence of mouse Zcytor16 ina biological sample can be tested by mixing the biological sample with atrace amount of labeled mouse Zcytor16 and an anti-mouse Zcytor16antibody under conditions that promote binding between mouse Zcytor16and its antibody. Complexes of mouse Zcytor16 and anti-mouse Zcytor16 inthe sample can be separated from the reaction mixture by contacting thecomplex with an immobilized protein which binds with the antibody, suchas an Fc antibody or Staphylococcus protein A. The concentration ofmouse Zcytor16 in the biological sample will be inversely proportionalto the amount of labeled mouse Zcytor16 bound to the antibody anddirectly related to the amount of free labeled mouse Zcytor16.Illustrative biological samples include blood, urine, saliva, tissuebiopsy, and autopsy material.

Alternatively, in vitro assays can be performed in which anti-mouseZcytor16 antibody is bound to a solid-phase carrier. For example,antibody can be attached to a polymer, such as aminodextran, in order tolink the antibody to an insoluble support such as a polymer-coated bead,a plate or a tube. Other suitable in vitro assays will be readilyapparent to those of skill in the art.

In another approach, anti-mouse Zcytor16 antibodies can be used todetect Zcytor16 in tissue sections prepared from a biopsy specimen. Suchimmunochemical detection can be used to determine the relative abundanceof mouse Zcytor16 and to determine the distribution of Zcytor16 in theexamined tissue. General immunochemistry techniques are well established(see, for example, Ponder, “Cell Marking Techniques and TheirApplication,” in Mammalian Development: A Practical Approach, Monk(ed.), pages 115-38 (IRL Press 1987), Coligan at pages 5.8.1-5.8.8,Ausubel (1995) at pages 14.6.1 to 14.6.13 (Wiley Interscience 1990), andManson (ed.), Methods In Molecular Biology, Vol. 10: ImmunochemicalProtocols (The Humana Press, Inc. 1992)).

Immunochemical detection can be performed by contacting a biologicalsample with an anti-mouse Zcytor16 antibody, and then contacting thebiological sample with a detectably labeled molecule which binds to theantibody. For example, the detectably labeled molecule can comprise anantibody moiety that binds to anti-mouse Zcytor16 antibody.Alternatively, the anti-mouse Zcytor16 antibody can be conjugated withavidin/streptavidin (or biotin) and the detectably labeled molecule cancomprise biotin (or avidin/streptavidin). Numerous variations of thisbasic technique are well-known to those of skill in the art.

Alternatively, an anti-mouse Zcytor16 antibody can be conjugated with adetectable label to form an anti-mouse Zcytor16 immunoconjugate.Suitable detectable labels include, for example, a radioisotope, afluorescent label, a chemiluminescent label, an enzyme label, abioluminescent label or colloidal gold. Methods of making and detectingsuch detectably-labeled immunoconjugates are well-known to those ofordinary skill in the art, and are described in more detail below.

The detectable label can be a radioisotope that is detected byautoradiography. Isotopes that are particularly useful for the purposeof the present invention are ³H, ¹²⁵I, ¹³¹I, ³⁵S and ¹⁴C.

Anti-mouse Zcytor16 immunoconjugates can also be labeled with afluorescent compound. The presence of a fluorescently-labeled antibodyis determined by exposing the immunoconjugate to light of the properwavelength and detecting the resultant fluorescence. Fluorescentlabeling compounds include fluorescein isothiocyanate, rhodamine,phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine.

Alternatively, anti-mouse Zcytor16 immunoconjugates can be detectablylabeled by coupling an antibody component to a chemiluminescentcompound. The presence of the chemiluminescent-tagged immunoconjugate isdetermined by detecting the presence of luminescence that arises duringthe course of a chemical reaction. Examples of chemiluminescent labelingcompounds include luminol, isoluminol, an aromatic acridinium ester, animidazole, an acridinium salt and an oxalate ester.

Similarly, a bioluminescent compound can be used to label anti-mouseZcytor16 immunoconjugates of the present invention. Bioluminescence is atype of chemiluminescence found in biological systems in which acatalytic protein increases the efficiency of the chemiluminescentreaction. The presence of a bioluminescent protein is determined bydetecting the presence of luminescence. Bioluminescent compounds thatare useful for labeling include luciferin, luciferase and aequorin.

Alternatively, anti-mouse Zcytor16 immunoconjugates can be detectablylabeled by linking an anti-mouse Zcytor16 antibody component to anenzyme. When the anti-mouse Zcytor16-enzyme conjugate is incubated inthe presence of the appropriate substrate, the enzyme moiety reacts withthe substrate to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorometric or visual means. Examplesof enzymes that can be used to detectably label polyspecificimmunoconjugates include β-galactosidase, glucose oxidase, peroxidaseand alkaline phosphatase.

Those of skill in the art will know of other suitable labels which canbe employed in accordance with the present invention. The binding ofmarker moieties to anti-mouse Zcytor16 antibodies can be accomplishedusing standard techniques known to the art. Typical methodology in thisregard is described by Kennedy et al., Clin. Chim. Acta 70:1 (1976),Schurs et al., Clin. Chim. Acta 81:1 (1977), Shih et al., Int'l J.Cancer 46:1101 (1990), Stein et al., Cancer Res. 50:1330 (1990), andColigan, supra.

Moreover, the convenience and versatility of immunochemical detectioncan be enhanced by using anti-mouse Zcytor16 antibodies that have beenconjugated with avidin, streptavidin, and biotin (see, for example,Wilchek et al. (eds.), “Avidin-Biotin Technology,” Methods InEnzymology, Vol. 184 (Academic Press 1990), and Bayer et al.,“Immunochemical Applications of Avidin-Biotin Technology,” in Methods InMolecular Biology, Vol. 10, Manson (ed.), pages 149-162 (The HumanaPress, Inc. 1992).

Methods for performing immunoassays are well-established. See, forexample, Cook and Self, “Monoclonal Antibodies in DiagnosticImmunoassays,” in Monoclonal Antibodies: Production, Engineering, andClinical Application, Ritter and Ladyman (eds.), pages 180-208,(Cambridge University Press, 1995), Perry, “The Role of MonoclonalAntibodies in the Advancement of Immunoassay Technology,” in MonoclonalAntibodies: Principles and Applications, Birch and Lennox (eds.), pages107-120 (Wiley-Liss, Inc. 1995), and Diamandis, Immunoassay (AcademicPress, Inc. 1996).

The present invention also contemplates kits for performing animmunological diagnostic assay for Zcytor16. gene expression. Such kitscomprise at least one container comprising an anti-mouse Zcytor16antibody, or antibody fragment. A kit may also comprise a secondcontainer comprising one or more reagents capable of indicating thepresence of mouse Zcytor16 antibody or antibody fragments. Examples ofsuch indicator reagents include detectable labels such as a radioactivelabel, a fluorescent label, a chemiluminescent label, an enzyme label, abioluminescent label, colloidal gold, and the like. A kit may alsocomprise a means for conveying to the user that mouse Zcytor16antibodies or antibody fragments are used to detect Zcytor16 protein.For example, written instructions may state that the enclosed antibodyor antibody fragment can be used to detect mouse Zcytor16.The writtenmaterial can be applied directly to a container, or the written materialcan be provided in the form of a packaging insert.

Alternative techniques for generating or selecting antibodies usefulherein include in vitro exposure of lymphocytes to soluble mouseZcytor16 monomeric receptor or soluble mouse Zcytor16 homodimeric,heterodimeric or multimeric polypeptides, and selection of antibodydisplay libraries in phage or similar vectors (for instance, through useof immobilized or labeled soluble mouse Zcytor16 monomeric receptor orsoluble mouse Zcytor16 homodimeric, heterodimeric or multimericpolypeptides). Genes encoding polypeptides having potential bindingdomains such as soluble mouse Zcytor16 monomeric receptor or solublemouse Zcytor16 homodimeric, heterodimeric or multimeric polypeptide canbe obtained by screening random peptide libraries displayed on phage(phage display) or on bacteria, such as E. coli. Nucleotide sequencesencoding the polypeptides can be obtained in a number of ways, such asthrough random mutagenesis and random polynucleotide synthesis. Theserandom peptide display libraries can be used to screen for peptideswhich interact with a known target which can be a protein orpolypeptide, such as a ligand or receptor, a biological or syntheticmacromolecule, or organic or inorganic substances. Techniques forcreating and screening such random peptide display libraries are knownin the art (Ladner et al., U.S. Pat. No. 5,223,409; Ladner et al., U.S.Pat. No. 4,946,778; Ladner et al., U.S. Pat. No. 5,403,484 and Ladner etal., U.S. Pat. No. 5,571,698) and random peptide display libraries andkits for screening such libraries are available commercially, forinstance from Clontech (Palo Alto, Calif.), Invitrogen Inc; (San Diego,Calif.), New England Biolabs, Inc. (Beverly, Mass.) and Pharmacia LKBBiotechnology Inc. (Piscataway, N.J.). Random peptide display librariescan be screened using the soluble mouse Zcytor16 monomeric receptor orsoluble mouse Zcytor16 homodimeric, heterodimeric or multimericpolypeptide sequences disclosed herein to identify proteins which bindto mouse Zcytor16-comprising receptor polypeptides. These “bindingpolypeptides,” which interact with soluble mouse Zcytor16-comprisingreceptor polypeptides, can be used for tagging cells, for example, thosein which Zcytor16 is expressed; for isolating homolog polypeptides byaffinity purification; they can be directly or indirectly conjugated todrugs, toxins, radionuclides and the like. These binding polypeptidescan also be used in analytical methods such as for screening expressionlibraries and neutralizing activity, e.g., for blocking interactionbetween IL-TIF ligand and receptor, or viral binding to a receptor. Thebinding polypeptides can also be used for diagnostic assays fordetermining circulating levels of soluble mouse Zcytor16-comprisingreceptor polypeptides; for detecting or quantitating soluble ornon-soluble Zcytor16-comprising receptors as marker of underlyingpathology or disease. These binding polypeptides can also act as“antagonists” to block soluble or membrane-bound mouse Zcytor16monomeric receptor or mouse Zcytor16 homodimeric, heterodimeric ormultimeric polypeptide binding (e.g. to ligand) and signal transductionin vitro and in vivo. Again, these binding polypeptides serve asanti-mouse Zcytor16 monomeric receptor or anti-mouse Zcytor16homodimeric, heterodimeric or multimeric polypeptides and are useful forinhibiting IL-TIF activity, as well as receptor activity orprotein-binding. Antibodies raised to the natural receptor complexes ofthe present invention may be preferred embodiments, as they may act morespecifically against the IL-TIF, or more potently than antibodies raisedto only one subunit. Moreover, the antagonistic and binding activity ofthe antibodies of the present invention can be assayed in the IL-TIFproliferation, signal trap, luciferase or binding assays in the presenceof IL-TIF and Zcytor16-comprising soluble receptors, and otherbiological or biochemical assays described herein.

Antibodies to monomeric mouse Zcytor16 receptor or mouse Zcytor16homodimeric, heterodimeric or multimeric mouse Zcytor16-containingreceptors may be used for tagging cells that express Zcytor16 receptors;for isolating soluble Zcytor16-comprising receptor polypeptides byaffinity purification; for diagnostic assays for determining circulatinglevels of soluble Zcytor16-comprising receptor polypeptides; fordetecting or quantitating soluble Zcytor16-comprising receptors asmarker of underlying pathology or disease; in analytical methodsemploying FACS; for screening expression libraries; for generatinganti-idiotypic antibodies that can act as IL-TIF agonists; and asneutralizing antibodies or as antagonists to block Zcytor16 receptorfunction, or to block IL-TIF activity in vitro and in vivo. Suitabledirect tags or labels include radionuclides, enzymes, substrates,cofactors, biotin, inhibitors, fluorescent markers, chemiluminescentmarkers, magnetic particles and the like; indirect tags or labels mayfeature use of biotin-avidin or other complement/anti-complement pairsas intermediates. Antibodies herein may also be directly or indirectlyconjugated to drugs, toxins, radionuclides and the like, and theseconjugates used for in vivo diagnostic or therapeutic applications.Moreover, antibodies to soluble mouse Zcytor16-comprising receptorpolypeptides, or fragments thereof may be used in vitro to detectdenatured or non-denatured mouse or orthologous Zcytor16-comprisingreceptor polypeptides or fragments thereof in assays, for example,Western Blots or other assays known in the art.

Antibodies to soluble mouse Zcytor16 receptor or soluble mouse Zcytor16homodimeric, heterodimeric or multimeric receptor polypeptides areuseful for tagging cells that express the corresponding receptors andassaying their expression levels, for affinity purification, withindiagnostic assays for determining circulating levels of receptorpolypeptides, analytical methods employing fluorescence-activated cellsorting. Moreover, divalent antibodies, and anti-idiotypic antibodiesmay be used as agonists to mimic the effect of the Zcytor16 ligand,IL-TIF.

Antibodies herein can also be directly or indirectly conjugated todrugs, toxins, radionuclides and the like, and these conjugates used forin vivo diagnostic or therapeutic applications. For instance, antibodiesor binding polypeptides which recognize soluble mouse Zcytor16 receptoror soluble mouse Zcytor16 homodimeric, heterodimeric or multimericreceptor polypeptides of the present invention can be used to identifyor treat tissues or organs that express a correspondinganti-complementary molecule (i.e., a mouse Zcytor16-comprising solubleor membrane-bound receptor). More specifically, antibodies to solublemouse, Zcytor16-comprising receptor polypeptides, or bioactive fragmentsor portions thereof, can be coupled to detectable or cytotoxic moleculesand delivered to a mammal having cells, tissues or organs that express aZcytor16-comprising receptor such as Zcytor16-expressing cancers, orcertain disease states.

Suitable detectable molecules may be directly or indirectly attached topolypeptides that bind mouse Zcytor16-comprising receptor polypeptides,such as “binding polypeptides,” (including binding peptides disclosedabove), antibodies, or bioactive fragments or portions thereof. Suitabledetectable molecules include radionuclides, enzymes, substrates,cofactors, inhibitors, fluorescent markers, chemiluminescent markers,magnetic particles and the like. Suitable cytotoxic molecules may bedirectly or indirectly attached to the polypeptide or antibody, andinclude bacterial or plant toxins (for instance, diphtheria toxin,Pseudomonas exotoxin, ricin, abrin and the like), as well as therapeuticradionuclides, such as iodine-131, rhenium-188 or yttrium-90 (eitherdirectly attached to the polypeptide or antibody, or indirectly attachedthrough means of a chelating moiety, for instance). Binding polypeptidesor antibodies may also be conjugated to cytotoxic drugs, such asadriamycin. For indirect attachment of a detectable or cytotoxicmolecule, the detectable or cytotoxic molecule can be conjugated with amember of a complementary/anticomplementary pair, where the other memberis bound to the binding polypeptide or antibody portion. For thesepurposes, biotin/streptavidin is an exemplarycomplementary/anticomplementary pair.

In another embodiment, binding polypeptide-toxin fusion proteins orantibody-toxin fusion proteins can be used for targeted cell or tissueinhibition or ablation (for instance, to treat cancer cells or tissues).Alternatively, if the binding polypeptide has multiple functionaldomains (i.e., an activation domain or a ligand binding domain, plus atargeting domain), a fusion protein including only the targeting domainmay be suitable for directing a detectable molecule, a cytotoxicmolecule or a complementary molecule to a cell or tissue type ofinterest. In instances where the fusion protein including only a singledomain includes a complementary molecule, the anti-complementarymolecule can be conjugated to a detectable or cytotoxic molecule. Suchdomain-complementary molecule fusion proteins thus represent a generictargeting vehicle for cell/tissue-specific delivery of genericanti-complementary-detectable/cytotoxic molecule conjugates.

In another embodiment, mouse Zcytor16 binding polypeptide-cytokine orantibody-cytokine fusion proteins can be used for enhancing in vivokilling of target tissues (for example, spleen, pancreatic, blood,lymphoid, colon, and bone marrow cancers), if the bindingpolypeptide-cytokine or anti-mouse Zcytor16 receptor antibody targetsthe hyperproliferative cell (See, generally, Hornick et al., Blood89:4437-47, 1997). The described fusion proteins enable targeting of acytokine to a desired site of action, thereby providing an elevatedlocal concentration of cytokine. Suitable anti-mouse Zcytor16 monomer,homodimer, heterodimer or multimer antibodies target an undesirable cellor tissue (i.e., a tumor or a leukemia), and the fused cytokine mediatesimproved target cell lysis by effector cells. Suitable cytokines forthis purpose include interleukin 2 and granulocyte-macrophagecolony-stimulating factor (GM-CSF), for instance.

Alternatively, mouse Zcytor16 receptor binding polypeptides or antibodyfusion proteins described herein can be used for enhancing in vivokilling of target tissues by directly stimulating a Zcytor16receptor-modulated apoptotic pathway, resulting in cell death ofhyperproliferative cells expressing Zcytor16-comprising receptors.

12. Production of Transgenic Mice

Mice engineered to overexpress the mouse Zcytor16 gene, referred to as“transgenic mice,” and mice that exhibit a complete absence of mouseZcytor16 gene function, referred to as “knockout mice,” may also begenerated (Snouwaert et al., Science 257:1083, 1992; Lowell et al.,Nature 366:740-42, 1993; Capecchi, M. R., Science 244: 1288-1292, 1989;Palmiter, R. D. et al. Annu Rev Genet. 20: 465-499, 1986). For example,transgenic mice that over-express mouse Zcytor16, either ubiquitously orunder a tissue-specific or tissue-restricted promoter can be used to askwhether over-expression causes a phenotype. For example, over-expressionof a wild-type Zcytor16 polypeptide, polypeptide fragment or a mutantthereof, may alter normal cellular processes, or repress function of theZcytor16 ligand IL-TIF, resulting in a phenotype that identifies atissue in which Zcytor16 expression is functionally relevant and mayindicate a therapeutic target for the Zcytor16, its agonists orantagonists. For example, a preferred transgenic mouse to engineer isone that over-expresses the full length human Zcytor16 polypeptide (SEQID NO:2); or more preferably the full length mouse Zcytor16 polypeptide(SEQ ID NO:38 or SEQ ID NO:48), or mature nouse polypeptide (Amino acids24 to 230, or 27 to 230 of SEQ ID NO:38 or SEQ ID NO:48). Preferredtissue-specific or tissue-restricted promoters include monocyte andlymphoid-restricted, epithelial-specific, lung-specific, ovary-specificand skin-restricted promoters. Moreover, such over-expression may resultin a phenotype that shows similarity with human diseases, or thatcounteracts the effects of increased or administered IL-TIF (IL-TIFadenovirus in mice suggests that IL-TIF over-expression increases thelevel of neutrophils and platelets in vivo).

Similarly, knockout mouse Zcytor16 mice can be used to determine whereZcytor16 is absolutely required in vivo. Such knockout animals serve asmodels to understand the requirements of Zcytor16 function in humans. Atransgenic mouse that is a knockout mouse would not expresses residue 1to 230 or 24 to 230, or 27 to 230 of SEQ ID NO:38 or SEQ ID NO:48,because they would exhibit a complete absence of endogenous Zcytor16gene function. The phenotype of knockout mice is predictive of the invivo effects of that a Zcytor16 antagonist, such as those describedherein, may have. The murine Zcytor16 mRNA, and cDNA is isolated (SEQ IDNO:37 or SEQ ID NO:47) and can be used to isolate mouse Zcytor16 genomicDNA, which are subsequently used to generate knockout mice. Thesetransgenic and knockout mice may be employed to study the Zcytor16 geneand the protein encoded thereby in an in vivo system, and can be used asin vivo models for corresponding human or animal diseases (such as thosein commercially viable animal populations). The mouse models of thepresent invention are particularly relevant as tumor models for thestudy of immune function, inflammatory disease, cancer biology andprogression. Such models are useful in the development and efficacy oftherapeutic molecules used in human cancers, immune and inflammatorydiseases. For example, because increases in human Zcytor16 expressionare associated with specific human cancers, such as ovarian cancer, bothtransgenic mice and knockout mice would serve as useful animal modelsfor cancer. Moreover, because increases in human Zcytor16 expression areassociated with specific monocyte cells, such CD4+ T-cells and CD19+B-cells, both mouse Zcytor16 transgenic mice and knockout mice wouldserve as useful animal models for immune function, inflammation, immunedisorders, infection, anemia, hematopoietic and other cancers. Moreover,because Zcytor16 is a receptor for IL-TIF, use of mouse Zcytor16transgenic and knockout micein a mouse model employing IL-TIF (e.g.,with and without IL-TIF) cans serve as an additional animal model tostudy the development and efficacy of therapeutic molecules used inhuman cancers, immune and inflammatory diseases associated with IL-TIF,as well as assess the efficacy and usefulness of IL-TIF antagonists invivo, such as the zcyr16 soluble receptors discussed herein. Moreover,in a preferred embodiment, Zcytor16 transgenic mouse can serve as ananimal model for specific tumors, particularly ovarian cancer, stomachcancer, uterine cancer, rectal cancer, lung cancer and esophagealcancer; and as a model for IL-TIF-induced inflammation where Zcytor16polypeptide is an antagonist. Such mouse zcytor16 transgenic andknowckout mouse models can also be used to assess the therapeuticaspects of IL-TIF, chemical therapeutics, anti-IL-TIF antibodies,anti-Zcytor16 antibodies, or Zcytor16 soluble receptors therein.Moreover, transgenic mice expression of mouse Zcytor16 antisensepolynucleotides or ribozymes directed against mouse Zcytor16, describedherein, can be used analogously to transgenic mice described above.

Transgenic mice can be engineered to over-express the mouse Zcytor16gene in all tissues or under the control of a tissue-specific ortissue-preferred regulatory element. These over-producers of mouseZcytor16 can be used to characterize the phenotype that results fromover-expression, and the transgenic animals can serve as models forhuman disease caused by excess mouse Zcytor16. Transgenic mice thatover-express mouse Zcytor16 also provide model bioreactors forproduction of mouse Zcytor16, such as soluble mouse Zcytor16, in themilk or blood of larger animals. Methods for producing transgenic miceare well-known to those of skill in the art (see, for example, Jacob,“Expression and Knockout of Interferons in Transgenic Mice,” inOverexpression and Knockout of Cytokines in Transgenic Mice, Jacob(ed.), pages 111-124 (Academic Press, Ltd. 1994), Monasterky and Robl(eds), Strategies in Transgenic Animal Science (ASM Press 1995), andAbbud and Nilson, “Recombinant Protein Expression in Transgenic Mice,”in Gene Expression Systems: Using Nature for the Art of Expression,Fernandez and Hoeffler (eds.), pages 367-397 (Academic Press, Inc.1999)).

For example, a method for producing a transgenic mouse that expresses amouse Zcytor16 gene can begin with adult, fertile males (studs) (B6C3f1,2-8 months of age (Taconic Farms, Germantown, N.Y.)), vasectomized males(duds) (B6D2f1, 2-8 months, (Taconic Farms)), prepubescent fertilefemales (donors) (B6C3f1, 4-5 weeks, (Taconic Farms)) and adult fertilefemales (recipients) (B6D2f1, 2-4 months, (Taconic Farms)). The donorsare acclimated for one week and then injected with approximately 8IU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma Chemical Company;St. Louis, Mo.) I.P., and 46-47 hours later, 8 IU/mouse of humanChorionic Gonadotropin (hCG (Sigma)) I.P. to induce superovulation.Donors are mated with studs subsequent to hormone injections. Ovulationgenerally occurs within 13 hours of hCG injection. Copulation isconfirmed by the presence of a vaginal plug the morning followingmating.

Fertilized eggs are collected under a surgical scope. The oviducts arecollected and eggs are released into urinanalysis slides containinghyaluronidase (Sigma). Eggs are washed once in hyaluronidase, and twicein Whitten's W640 medium (described, for example, by Menino andO'Claray, Biol. Reprod. 77:159 (1986), and Dienhart and Downs, Zygote4:129 (1996)) that has been incubated with 5% CO₂, 5% O₂, and 90% N₂ at37° C. The eggs are then stored in a 37° C./5% CO₂ incubator untilmicroinjection.

Ten to twenty micrograms of plasmid DNA containing a mouse Zcytor16encoding sequence is linearized, gel-purified, and resuspended in 10 mMTris-HCl (pH 7.4), 0.25 mM EDTA (pH 8.0), at a final concentration of5-10 nanograms per microliter for microinjection. For example, the mouseZcytor16 encoding sequences can encode a polypeptide comprising theamino acid sequence of SEQ ID NO:38 or SEQ ID NO:48.

Plasmid DNA is microinjected into harvested eggs contained in a drop ofW640 medium overlaid by warm, CO₂-equilibrated mineral oil. The DNA isdrawn into an injection needle (pulled from a 0.75 mm ID, 1 mm ODborosilicate glass capillary), and injected into individual eggs. Eachegg is penetrated with the injection needle, into one or both of thehaploid pronuclei.

Picoliters of DNA are injected into the pronuclei, and the injectionneedle withdrawn without coming into contact with the nucleoli. Theprocedure is repeated until all the eggs are injected. Successfullymicroinjected eggs are transferred into an organ tissue-culture dishwith pre-gassed W640 medium for storage overnight in a 37° C./5% COincubator.

The following day, two-cell embryos are transferred into pseudopregnantrecipients. The recipients are identified by the presence of copulationplugs, after copulating with vasectomized duds. Recipients areanesthetized and shaved on the dorsal left side and transferred to asurgical microscope. A small incision is made in the skin and throughthe muscle wall in the middle of the abdominal area outlined by theribcage, the saddle, and the hind leg, midway between knee and spleen.The reproductive organs are exteriorized onto a small surgical drape.The fat pad is stretched out over the surgical drape, and a babyserrefine (Roboz, Rockville, Md.) is attached to the fat pad and lefthanging over the back of the mouse, preventing the organs from slidingback in.

With a fine transfer pipette containing mineral oil followed byalternating W640 and air bubbles, 12-17 healthy two-cell embryos fromthe previous day's injection are transferred into the recipient. Theswollen ampulla is located and holding the oviduct between the ampullaand the bursa, a nick in the oviduct is made with a 28 g needle close tothe bursa, making sure not to tear the ampulla or the bursa.

The pipette is transferred into the nick in the oviduct, and the embryosare blown in, allowing the first air bubble to escape the pipette. Thefat pad is gently pushed into the peritoneum, and the reproductiveorgans allowed to slide in. The peritoneal wall is closed with onesuture and the skin closed with a wound clip. The mice recuperate on a37° C. slide warmer for a minimum of four hours.

The recipients are returned to cages in pairs, and allowed 19-21 daysgestation. After birth, 19-21 days postpartum is allowed before weaning.The weanlings are sexed and placed into separate sex cages, and a 0.5 cmbiopsy (used for genotyping) is snipped off the tail with cleanscissors.

Genomic DNA is prepared from the tail snips using, for example, a QIAGENDNEASY kit following the manufacturer's instructions. Genomic DNA isanalyzed by PCR using primers designed to amplify a mouse Zcytor16 geneor a selectable marker gene that was introduced in the same plasmid.After animals are confirmed to be transgenic, they are back-crossed intoan inbred strain by placing a transgenic female with a wild-type male,or a transgenic male with one or two wild-type female(s). As pups areborn and weaned, the sexes are separated, and their tails snipped forgenotyping.

To check for expression of a transgene in a live animal, a partialhepatectomy is performed. A surgical prep is made of the upper abdomendirectly below the zyphoid process. Using sterile technique, a small1.5-2 cm incision is made below the sternum and the left lateral lobe ofthe liver exteriorized. Using 4-0 silk, a tie is made around the lowerlobe securing it outside the body cavity. An atraumatic clamp is used tohold the tie while a second loop of absorbable Dexon (American Cyanamid;Wayne, N.J.) is placed proximal to the first tie. A distal cut is madefrom the Dexon tie and approximately 100 mg of the excised liver tissueis placed in a sterile petri dish. The excised liver section istransferred to a 14 ml polypropylene round bottom tube and snap frozenin liquid nitrogen and then stored on dry ice. The surgical site isclosed with suture and wound clips, and the animal's cage placed on a37° C. heating pad for 24 hours post operatively. The animal is checkeddaily post operatively and the wound clips removed 7-10 days aftersurgery. The expression level of mouse Zcytor16 mRNA is examined foreach transgenic mouse using an RNA solution hybridization assay orpolymerase chain reaction.

In addition to producing transgenic mice that over-express mouseZcytor16, it is useful to engineer transgenic mice with eitherabnormally low or no expression of the gene. Such transgenic miceprovide useful models for diseases associated with a lack of mouseZcytor16. As discussed above, mouse Zcytor16 gene expression can beinhibited using anti-sense genes, ribozyme genes, or external guidesequence genes. To produce transgenic mice that under-express the mouseZcytor16 gene, such inhibitory sequences are targeted to mouse Zcytor16mRNA. Methods for producing transgenic mice that have abnormally lowexpression of a particular gene are known to those in the art (see, forexample, Wu et al., “Gene Underexpression in Cultured Cells and Animalsby Antisense DNA and RNA Strategies,” in Methods in Gene Biotechnology,pages 205-224 (CRC Press 1997)).

An alternative approach to producing transgenic mice that have little orno mouse Zcytor16 gene expression is to generate mice having at leastone normal mouse Zcytor16 allele replaced by a nonfunctional mouseZcytor16 gene. One method of designing a nonfunctional mouse Zcytor16gene is to insert another gene, such as a selectable marker gene, withina nucleic acid molecule that encodes mouse Zcytor16. Standard methodsfor producing these so-called “knockout mice” are known to those skilledin the art (see, for example, Jacob, “Expression and Knockout ofInterferons in Transgenic Mice,” in Overexpression and Knockout ofCytokines in Transgenic Mice, Jacob (ed.), pages 111-124 (AcademicPress, Ltd. 1994), and Wu et al., “New Strategies for Gene Knockout,” inMethods in Gene Biotechnology, pages 339-365 (CRC Press 1997)).

13. Therapeutic Uses of Polypeptides Having Zcytor16 Activity

Amino acid sequences having mouse Zcytor16 activity can be used tomodulate the immune system by binding Zcytor16 ligand, and thus,preventing the binding of Zcytor16 ligand with endogenous Zcytor16receptor. Zcytor16 antagonists, such as anti-mouse Zcytor16 antibodies,can also be used to modulate the immune system by inhibiting the bindingof Zcytor16 ligand with the endogenous Zcytor16 receptor. Accordingly,the present invention includes the use of proteins, polypeptides, andpeptides having mouse Zcytor16 activity (such as mouse Zcytor16polypeptides, mouse Zcytor16 analogs (e.g., anti-mouse Zcytor16anti-idiotype antibodies), and mouse Zcytor16 fusion proteins) to asubject which lacks an adequate amount of this polypeptide, or whichproduces an excess of Zcytor16 ligand. Mouse Zcytor16 antagonists (e.g.,anti-mouse Zcytor16 antibodies) can be also used to treat a subject thatproduces an excess of either Zcytor16, ligand or Zcytor16 receptor.Suitable subjects include mammals, such as humans. In addition, humanZcytor16 and IL-TIF activity described herein emphasizes the usefulnessof using mouse Zcytor16 in animal models, such as a mouse modeldescribed above for studying human inflammation, inflammatory diseases,immune function and diseases and cancer, or assessing therapeuticaspects of IL-TIF, chemical therapeutics, anti-IL-TIF antibodies,anti-Zcytor16 antibodies, or Zcytor16 soluble receptors therein.

Moreover, we have shown that the human Zcytor16 receptor binds a ligandcalled T-cell inducible Factor (IL-TIF) (SEQ ID NO:15) ; Dumoutier, L.et al., Proc. Nat'l. Acad. Sci. 97:10144-10149, 2000; mouse IL-TIFsequence (SEQ ID NO:41) is shown in Dumontier et al., J. Immunol.164:1814-1819, 2000). Moreover, commonly owned zcytor11 (U.S. Pat. No.5,965,704) and CRF2-4 receptor also bind IL-TIF (See, WIPO publicationWO 00/24758; Dumontier et al., J. Immunol. 164:1814-1819, 2000; Spencer,S D et al., J. Exp. Med. 187:571-578, 1998; Gibbs, V C and Pennica Gene186:97-101, 1997 (CRF2-4 cDNA); Xie, M H et al., J. Biol. Chem. 275:31335-31339, 2000; and Kotenko, S V et al., J. Biol. Chem. manuscript inpress M007837200). Moreover, IL-10β receptor may be involved as areceptor for IL-TIF, and it is believed to be synonymous with CRF2-4(Dumoutier, L. et al., Proc. Nat'l. Acad. Sci. 97:10144-10149, 2000; LiuY et al, J Immunol. 152; 1821-1829, 1994 (IL-10R cDNA). Within preferredembodiments, the soluble receptor form of mouse Zcytor16, residues24-230 of SEQ ID NO:38 or SEQ ID NO:48 is a monomer, homodimer,heterodimer, or multimer that antagonizes the effects of IL-TIF in vivo.Antibodies and binding polypeptides to such mouse Zcytor16 monomer,homodimer, heterodimer, or multimers also serve as antagonists ofZcytor16 activity.

IL-TIF has been shown to be induced in the presence of IL-9, and issuspected to be involved in promoting Th1-type immune responses, andinflammation. IL-9 stimulates proliferation, activation, differentiationand/or induction of immune function in a variety of ways and isimplicated in asthma, lung mastocytosis, and other diseases, as well asactivates STAT pathways. Antagonists of IL-TIF or IL-9 function can havebeneficial use against such human diseases. The present inventionprovides such novel antagonists of IL-TIF.

IL-TIF has been show to be involved in up-regulate the production ofacute phase reactants, such as serum amyloid A (SAA),α1-antichymotrypsin, and haptoglobin, and that IL-TIF expression isincreased upon injection of lipopolysaccharide (LPS) in vivo suggestingthat IL-TIF is involved in inflammatory response (Dumoutier, L. et al.,Proc. Nat'l. Acad. Sci. 97:10144-10149, 2000). Production of acute phaseproteins, such as SAA, is considered s short-term survival mechanismwhere inflammation is beneficial; however, maintenance of acute phaseproteins for longer periods contributes to chronic inflammation and canbe harmful to human health. For review, see Uhlar, CM and Whitehead, AS, Eur. J. Biochem. 265:501-523, 1999, and Baumann H. and Gauldie, J.Immunology Today 15:74-80, 1994. Moreover, the acute phase protein SAAis implicated in the pathogenesis of several chronic inflammatorydiseases, is implicated in atherosclerosis and rheumatoid arthritis, andis the precursor to the amyloid A protein deposited in amyloidosis(Uhlar, CM and Whitehead, supra.). Thus, as IL-TIF acts as apro-inflammatory molecule and induces production of SAA, antagonistswould be useful in treating inflammatory disease and other diseasesassociated with acute phase response proteins induced by IL-TIF. Suchantagonists are provided by the present invention. For example, methodof reducing IL-TIF-induced or IL-9 induced inflammation comprisesadministering to a mammal with inflammation an amount of a compositionof soluble mouse Zcytor16-comprising receptor sufficient to reduceinflammation. Moreover, a method of suppressing an inflammatory responsein a mammal with inflammation can comprise: (1) determining a level ofserum amyloid A protein; (2) administering a composition comprising asoluble mouse Zcytor16 cytokine receptor polypeptide as described hereinin an acceptable pharmaceutical vehicle; (3) determining a postadministration level of serum amyloid A protein; (4) comparing the levelof serum amyloid A protein in step (1) to the level of serum amyloid Aprotein in step (3), wherein a lack of increase or a decrease in serumamyloid A protein level is indicative of suppressing an inflammatoryresponse.

The receptors of the present invention include at least one mouseZcytor16 receptor subunit. A second receptor polypeptide included in theheterodimeric soluble receptor belongs to the receptor subfamily thatincludes Interleukin-10 receptor, the interferons (e.g.,interferon-gamma alpha and beta chains and theinterferon-alpha/betareceptor alpha and beta chains), zcytor7, zcytor11,and CRF2-4. A second soluble receptor polypeptide included in aheterodimeric soluble receptor can also include a zcytor11 solublereceptor subunit, disclosed in the commonly owned U.S. Pat. No.5,965,704; an EL-10R subunit, such as EL-10Rα; or a zcytor7 solublereceptor subunit, disclosed in the commonly owned U.S. Pat. No.5,945,511. The zcytor11 receptor in conjunction with CRF2-4 and IL-10Receptor was shown to signal JAK-STAT pathway in response to IL-TIF (Xieet al., supra.; Kotenko et al., supra.). According to the presentinvention, in addition to a monomeric or homodimeric mouse Zcytor16receptor polypeptide, a heterodimeric soluble mouse Zcytor16 receptor asexemplified by an embodiment comprising a soluble mouse Zcytor16receptor+soluble CRF2-4 receptor heterodimer (mouse Zcytor16/CRF2-4),can act as an antagonist of the IL-TIF. Other embodiments includesoluble heterodimers comprising mouse Zcytor16/IL-10R, mouseZcytor16/IL-9R, mouse Zcytor16/zcytor11, mouse Zcytor16/zcytor7, andother class II receptor subunits, as well as multimeric receptorsincluding but not limited to mouse Zcytor16/CRF2-4/zcytor11 or mouseZcytor16/CRF2-4/]L-10R.

Analysis of the tissue distribution of the mRNA corresponding mouseZcytor16 cDNA was similar to human Zcytor16 tissue distribution (U.S.patent application Ser. No. 09/728,911), and showed that mRNA wasespressed in placenta and spleen, and the ligand to which Zcytor16 binds(IL-TIF) is implicated in inducing inflammatory response includinginduction of the acute-phase response (Dumoutier, L. et al., Proc.Nat'l. Acad. Sci. 97:10144-10149, 2000). Thus, particular embodiments ofthe present invention are directed toward use of soluble mouse Zcytor16heterodimers as antagonists in inflammatory and immune diseases orconditions such as pancreatitis, type I diabetes (IDDM), pancreaticcancer, pancreatitis, Graves Disease, inflammatory bowel disease (IBD),Crohn's Disease, colon and intestinal cancer, diverticulosis, autoimmunedisease, sepsis, organ or bone marrow transplant; inflammation due totrauma, sugery or infection; amyloidosis; splenomegaly; graft versushost disease; and where inhibition of inflammation, immune suppression,reduction of proliferation of hematopoietic, immune, inflammatory orlymphoid cells, macrophages, T-cells (including Th1 and Th2 cells),suppression of immune response to a pathogen or antigen, or otherinstances where inhibition of IL-TIF or IL-9 cytokine production isdesired.

Moreover, antibodies or binding polypeptides that bind mouse Zcytor16polypeptides, monomers, homodimers, heterodimers and multimers describedherein and/or mouse Zcytor16 polypeptides, monomers, homodimers,heterodimers and multimers themselves are useful to:

-   -   1) Antagonize or block signaling via the IL-TIF receptors in the        treatment of acute inflammation, inflammation as a result of        trauma, tissue injury, surgery, sepsis or infection, and chronic        inflammatory diseases such as asthma, inflammatory bowel disease        (IBD), chronic colitis, splenomegaly, rheumatoid arthritis,        recurrent acute inflammatory episodes (e.g., tuberculosis), and        treatment of amyloidosis, and atherosclerosis, Castleman's        Disease, asthma, and other diseases associated with the        induction of acute-phase response.    -   2) Antagonize or block signaling via the IL-TIF receptors in the        treatment of autoimmune diseases such as IDDM, multiple        sclerosis (MS), systemic Lupus erythematosus (SLE), myasthenia        gravis, rheumatoid arthritis, and IBD to prevent or inhibit        signaling in immune cells (e.g. lymphocytes, monocytes,        leukocytes) via mouse Zcytor16 (Hughes C et al., J. Immunol 153:        3319-3325, 1994). Alternatively antibodies, such as monoclonal        antibodies (MAb) to mouse Zcytor16-comprising receptors, can        also be used as an antagonist to deplete unwanted immune cells        to treat autoimmune disease. Asthma, allergy and other atopic        disease may be treated with an MAb against, for example, soluble        mouse Zcytor16 soluble receptors or mouse Zcytor16/CRF2-4        heterodimers, to inhibit the immune response or to deplete        offending cells. Blocking or inhibiting signaling via Zcytor16,        using the polypeptides and antibodies of the present invention,        may also benefit diseases of the pancreas, kidney, pituitary and        neuronal cells. IDDM, NIDDM, pancreatitis, and pancreatic        carcinoma may benefit. Mouse Zcytor16 may serve as a target for        MAb therapy of cancer where an antagonizing MAb inhibits cancer        growth and targets immune-mediated killing. (Holliger, P, and        Hoogenboom, H: Nature Biotech. 16: 1015-1016, 1998). Mabs to        soluble mouse Zcytor16 monomer, homodimers, heterodimers and        multimers may also be useful to treat nephropathies such as        glomerulosclerosis, membranous neuropathy, amyloidosis (which        also affects the kidney among other tissues), renal        arteriosclerosis, glomerulonephritis of various origins,        fibroproliferative diseases of the kidney, as well as kidney        dysfunction associated with SLE, IDDM, type II diabetes (NIDDM),        renal tumors and other diseases.    -   3) Agonize or initiate signaling via the IL-TIF receptors in the        treatment of autoimmune diseases such as IDDM, MS, SLE,        myasthenia gravis, rheumatoid arthritis, and IBD. Anti-soluble        mouse Zcytor16, anti-soluble mouse Zcytor16/CRF2-4 heterodimers        and multimer monoclonal antibodies may signal lymphocytes or        other immune cells to differentiate, alter proliferation, or        change production of cytokines or cell surface proteins that        ameliorate autoimmunity. Specifically, modulation of a T-helper        cell response to an alternate pattern of cytokine secretion may        deviate an autoimmune response to ameliorate disease (Smith J A        et al., J. Immunol. 160:4841-4849, 1998). Similarly, agonistic        Anti-soluble mouse Zcytor16, anti-solublemouse Zcytor16/CRF2-4        heterodimers and multimer monoclonal antibodies may be used to        signal, deplete and deviate immune cells involved in asthma,        allergy and atopoic disease. Signaling via mouse Zcytor16 may        also benefit diseases of the pancreas, kidney, pituitary and        neuronal cells. IDDM, NIDDM, pancreatitis, and pancreatic        carcinoma may benefit. mouse Zcytor16 may serve as a target for        MAb therapy of pancreatic cancer where a signaling MAb inhibits        cancer growth and targets immune-mediated killing (Tutt, A L et        al., J Immunol. 161: 3175-3185, 1998). Similarly renal cell        carcinoma may be treated with monoclonal antibodies to mouse        Zcytor16-comprising soluble receptors of the present invention.

Soluble mouse Zcytor16 monomeric, homodimeric, heterodimeric andmultimeric polypeptides described herein can be used to neutralize/blockIL-TIF activity in the treatment of autoimmune disease, atopic disease,NIDDM, pancreatitis and kidney dysfunction as described above. A solubleform of mouse Zcytor16 may be used to promote an antibody responsemediated by Th cells and/or to promote the production of IL-4 or othercytokines by lymphocytes or other immune cells.

The soluble mouse Zcytor16-comprising receptors of the present inventionare useful as antagonists of the IL-TIF cytokine. Such antagonisticeffects can be achieved by direct neutralization or binding of theIL-TIF. In addition to antagonistic uses, the soluble receptors of thepresent invention can bind IL-TIF and act as carrier proteins for theIL-TIF cytokine, in order to transport the Ligand to different tissues,organs, and cells within the body. As such, the soluble receptors of thepresent invention can be fused or coupled to molecules, polypeptides orchemical moieties that direct the soluble-receptor-Ligand complex to aspecific site, such as a tissue, specific immune cell, or tumor. Forexample, in acute infection or some cancers, benefit may result frominduction of inflammation and local acute phase response proteins by theaction of IL-TIF. Thus, the soluble receptors of the present inventioncan be used to specifically direct the action of the IL-TIF. See,Cosman, D. Cytokine 5: 95-106, 1993; and Fernandez-Botran, R. Exp. Opin.Invest. Drugs 9:497-513, 2000.

Moreover, the soluble receptors of the present invention can be used tostabilize the IL-TIF, to increase the bioavailability, therapeuticlongevity, and/or efficacy of the Ligand by stabilizing the Ligand fromdegradation or clearance, or by targeting the ligand to a site of actionwithin the body. For example the naturally occurring IL-6/soluble IL-6Rcomplex stabilizes IL-6 and can signal through the gp130 receptor. See,Cosman, D. supra., and Fernandez-Botran, R. supra. Moreover, mouseZcytor16 may be combined with a cognate ligand such as IL-TIF tocomprise a ligand/soluble receptor complex. Such complexes may be usedto stimulate responses from cells presenting a companion receptorsubunit such as, for example, zcytor11 or CRF2-4. The cell specificityof mouse Zcytor16/ligand complexes may differ from that seen for theligand administered alone. Furthermore the complexes may have distinctpharmacokinetic properties such as affecting half-life, dose/responseand organ or tissue specificity. ZcytoR16/IL-TIF complexes thus may haveagonist activity to enhance an immune response or stimulate mesangialcells or to stimulate hepatic cells. Alternatively only tissuesexpressing a signaling subunit the heterodimerizes with the complex maybe affected analogous to the response to IL6/IL6R complexes (Hirota H.et al., Proc. Nat'l. Acad. Sci. 92:4862-4866, 1995; Hirano, T. inThomason, A. (Ed.) “The Cytokine Handbook”, 3^(rd) Ed., p. 208-209).Soluble receptor/cytokine complexes for IL12 and CNTF display similaractivities.

Mouse Zcytor16 homodimeric, heterodimeric and multimeric receptorpolypeptides may also be used within diagnostic systems for thedetection of circulating levels of IL-TIF ligand, and in the detectionof IL-TIF associated with acute phase inflammatory response. Within arelated embodiment, antibodies or other agents that bind to mouseZcytor16 soluble receptors of the present invention can be used todetect circulating receptor polypeptides; conversely, mouse Zcytor16soluble receptors themselves can be used to detect circulating orlocally-acting IL-TIF polypeptides. Elevated or depressed levels ofligand or receptor polypeptides may be indicative of pathologicalconditions, including inflammation or cancer. IL-TIF is known to induceassociated acute phase inflammatory response. Moreover, detection ofacute phase proteins or molecules such as IL-TIF can be indicative of achronic inflammatory condition in certain disease states (e.g.,rheumatoid arthritis). Detection of such conditions serves to aid indisease diagnosis as well as help a physician in choosing propertherapy.

Moreover, soluble mouse Zcytor16 receptor polypeptides of the presentinvention can be used as a “ligand sink,” i.e., antagonist, to bindligand in vivo or in vitro in therapeutic or other applications wherethe presence of the ligand is not desired. For example, in chronicinflammatory conditions or cancers that are expressing large amounts ofbioactive IL-TIF, soluble mouse Zcytor16 receptor or soluble mouseZcytor16 heterodimeric and multimeric receptor polypeptides, such assoluble mouse Zcytor16/CRF2-4 can be used as a direct antagonist of theligand in vivo, and may aid in reducing progression and symptomsassociated with the disease, and can be used in conjunction with othertherapies (e.g., steroid or chemotherapy) to enhance the effect of tothe therapy in reducing progression and symptoms, and preventingrelapse. Moreover, soluble mouse Zcytor16 receptor polypeptides can beused to slow the progression of cancers that over-express Zcytor16receptors, by binding ligand in vivo that could otherwise enhanceproliferation of those cancers.

Moreover, soluble mouse Zcytor16 receptor polypeptides of the presentinvention can be used in vivo or in diagnostic applications to detectIL-TIF-expressing inflammation or cancers in vivo or in tissue samples.For example, the soluble mouse Zcytor 16 receptors of the presentinvention can be conjugated to a radio-label or fluorescent label asdescribed herein, and used to detect the presence of the IL-TIF in atissue sample using an in vitro ligand-receptor type binding assay, orfluorescent imaging assay. Moreover, radiolabeled soluble mouse Zcytor16receptors of the present invention could be administered in vivo todetect Ligand-expressing solid tumors through a radio-imaging methodknown in the art.

Generally, the dosage of administered mouse Zcytor16 (or mouse Zcytor16analog or fusion protein) will vary depending upon such factors as thepatient's age, weight, height, sex, general medical condition andprevious medical history. Typically, it is desirable to provide therecipient with a dosage of mouse Zcytor16 polypeptide which is in therange of from about 1 pg/kg to 10 mg/kg (amount of agent/body weight ofpatient), although a lower or higher dosage also may be administered ascircumstances dictate.

Administration of a mouse Zcytor16 polypeptide to a subject can beintravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, intrapleural, intrathecal, by perfusion through a regionalcatheter, or by direct intralesional injection. When administeringtherapeutic proteins by injection, the administration may be bycontinuous infusion or by single or multiple boluses.

Additional routes of administration include oral, mucosal-membrane,pulmonary, and transcutaneous. Oral delivery is suitable for polyestermicrospheres, zein microspheres, proteinoid microspheres,polycyanoacrylate microspheres, and lipid-based systems (see, forexample, DiBase and Morrel, “Oral Delivery of MicroencapsulatedProteins,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 255-288 (Plenum Press 1997)). The feasibility of anintranasal delivery is exemplified by such a mode of insulinadministration (see, for example, Hinchcliffe and Illum, Adv. DrugDeliv. Rev. 35:199 (1999)). Dry or liquid particles comprising mouseZcytor16 can be prepared and inhaled with the aid of dry-powderdispersers, liquid aerosol generators, or nebulizers (e.g., Pettit andGombotz, TIBTECH 16:343 (1998); Patton et al., Adv. Drug Deliv. Rev.35:235 (1999)). This approach is illustrated by the AERX diabetesmanagement system, which is a hand-held electronic inhaler that deliversaerosolized insulin into the lungs. Studies have shown that proteins aslarge as 48,000 kDa have, been delivered across skin at therapeuticconcentrations with the aid of low-frequency ultrasound, whichillustrates the feasibility of trascutaneous administration (Mitragotriet al., Science 269:850 (1995)). Transdermal delivery usingelectroporation provides another means to administer a molecule havingmouse Zcytor16 binding activity (Potts et al., Pharm. Biotechnol. 10:213(1997)).

A pharmaceutical composition comprising a protein, polypeptide, orpeptide having mouse Zcytor16 binding activity can be formulatedaccording to known methods to prepare pharmaceutically usefulcompositions, whereby the therapeutic proteins are combined in a mixturewith a pharmaceutically acceptable carrier. A composition is said to bea “pharmaceutically acceptable carrier” if its administration can betolerated by a recipient patient. Sterile phosphate-buffered saline isone example of a pharmaceutically acceptable carrier. Other suitablecarriers are well-known to those in the art. See, for example, Gennaro(ed.), Remington's Pharmaceutical Sciences, 19th Edition (MackPublishing Company 1995).

For purposes of therapy, molecules having mouse Zcytor16 bindingactivity and a pharmaceutically acceptable carrier are administered to apatient in a therapeutically effective amount. A combination of aprotein, polypeptide, or peptide having mouse Zcytor16 binding activityand a pharmaceutically acceptable carrier is said to be administered ina “therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient patient. For example, an agent used to treat inflammation isphysiologically significant if its presence alleviates the inflammatoryresponse.

A pharmaceutical composition comprising mouse Zcytor16 (or mouseZcytor16 analog or fusion protein) can be furnished in liquid form, inan aerosol, or in solid form. Liquid forms, are illustrated byinjectable solutions and oral suspensions. Exemplary solid forms includecapsules, tablets, and controlled-release forms. The latter form isillustrated by miniosmotic pumps and implants (Bremer et al., Pharm.Biotechnol. 10:239 (1997); Ranade, “Implants in Drug Delivery,” in DrugDelivery Systems, Ranade and Hollinger (eds.), pages 95-123 (CRC Press1995); Bremer et al., “Protein Delivery with Infusion Pumps,”in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 239-254(Plenum Press 1997); Yewey et al., “Delivery of Proteins from aControlled Release Injectable Implant,” in Protein Delivery: PhysicalSystems, Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)).

Liposomes provide one means to deliver therapeutic polypeptides to asubject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.Infect. Dis. 12 (Suppl. 1):S61 (1993), Kim, Drugs 46:618 (1993), andRanade, “Site-Specific Drug Delivery Using Liposomes as Carriers,” inDrug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRCPress 1995)). Liposomes are similar in composition to cellular membranesand as a result, liposomes can be administered safely and arebiodegradable. Depending on the method of preparation, liposomes may beunilamellar or multilamellar, and liposomes can vary in size withdiameters ranging from 0.02 μm to greater than 10 μm. A variety ofagents can be encapsulated in liposomes: hydrophobic agents partition inthe bilayers and hydrophilic agents partition within the inner aqueousspace(s) (see, for example, Machy et al., Liposomes In Cell Biology AndPharmacology (John Libbey 1987), and Ostro et al., American J. Hosp.Pharm. 46:1576 (1989)). Moreover, it is possible to control thetherapeutic availability of the encapsulated agent by varying liposomesize, the number of bilayers, lipid composition, as well as the chargeand surface characteristics of the liposomes.

Liposomes can adsorb to virtually any type of cell and then slowlyrelease the encapsulated agent. Alternatively, an absorbed liposome maybe endocytosed by cells that are phagocytic. Endocytosis is followed byintralysosomal degradation of liposomal lipids and release of theencapsulated agents (Scherphof et al., Ann. N.Y. Acad. Sci. 446:368(1985)). After intravenous administration, small liposomes (0.1 to 1.0μm) are typically taken up by cells of the reticuloendothelial system,located principally in the liver and spleen, whereas liposomes largerthan 3.0 μm are deposited in the lung. This preferential uptake ofsmaller liposomes by the cells of the reticuloendothelial system hasbeen used to deliver chemotherapeutic agents to macrophages and totumors of the liver.

The reticuloendothelial system can be circumvented by several methodsincluding saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means (Claassen etal., Biochim. Biophys. Acta 802:428 (1984)). In addition, incorporationof glycolipid- or polyethelene glycol-derivatized phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system (Allen et al., Biochim.Biophys. Acta 1068:133 (1991); Allen et al., Biochim. Biophys. Acta1150:9 (1993)).

Liposomes can also be prepared to target particular cells or organs byvarying phospholipid composition or by inserting receptors or ligandsinto the liposomes. For example, liposomes, prepared with a high contentof a nonionic surfactant, have been used to target the liver (Hayakawaet al., Japanese Patent 04-244,018; Kato et al., Biol. Pharm. Bull.16:960 (1993)). These formulations were prepared by mixing soybeanphospatidylcholine, α-tocopherol, and ethoxylated hydrogenated castoroil (HCO-60) in methanol, concentrating the mixture under vacuum, andthen reconstituting the mixture with water. A liposomal formulation ofdipalmitoylphosphatidylcholine (DPPC) with a soybean-derivedsterylglucoside mixture (SG) and cholesterol (Ch) has also been shown totarget the liver (Shimizu et al., Biol. Pharm. Bull. 20:881 (1997)).

Alternatively, various targeting ligands can be bound to the surface ofthe liposome, such as antibodies, antibody fragments, carbohydrates,vitamins, and transport proteins. For example, liposomes can be modifiedwith branched type galactosyllipid derivatives to targetasialoglycoprotein (galactose) receptors, which are exclusivelyexpressed on the surface of liver cells (Kato and Sugiyama, Crit. Rev.Ther. Drug Carrier Syst. 14:287 (1997); Murahashi et al., Biol. Pharm.Bull.20:259 (1997)). Similarly, Wu et al., Hepatology 27:772 (1998),have shown that labeling liposomes with asialofetuin led to a shortenedliposome plasma half-life and greatly enhanced uptake ofasialofetuin-labeled liposome by hepatocytes. On the other hand, hepaticaccumulation of liposomes comprising branched type galactosyllipidderivatives can be inhibited by preinjection of asialofetuin (Murahashiet al., Biol. Pharm. Bull.20:259 (1997)). Polyaconitylated human serumalbum liposomes provide another approach for targeting liposomes toliver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681 (1997)).Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe ahepatocyte-directed liposome vesicle delivery system, which hasspecificity for hepatobiliary receptors associated with the specializedmetabolic cells of the liver.

In a more general approach to tissue targeting, target cells areprelabeled with biotinylated antibodies specific for a ligand expressedby the target cell (Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).After plasma elimination of free antibody, streptavidin-conjugatedliposomes are administered. In another approach, targeting antibodiesare directly attached to liposomes (Harasym et al., Adv. Drug Deliv.Rev. 32:99 (1998)).

Polypeptides having mouse Zcytor16 binding activity can be encapsulatedwithin liposomes using standard techniques of protein microencapsulation(see, for example, Anderson et al., Infect. Immun. 31:1099 (1981),Anderson et al., Cancer Res. 50:1853 (1990), and Cohen et al., Biochim.Biophys. Acta 1063:95 (1991), Alving et al. “Preparation and Use ofLiposomes in Immunological Studies,” in Liposome Technology, 2ndEdition, Vol. III, Gregoriadis (ed.), page 317 (CRC Press 1993), Wassefet al., Meth. Enzymol. 149:124 (1987)). As noted above, therapeuticallyuseful liposomes may contain a variety of components. For example,liposomes may comprise lipid derivatives of poly(ethylene glycol) (Allenet al., Biochim. Biophys. Acta 1150:9 (1993)).

Degradable polymer microspheres have been designed to maintain highsystemic levels of therapeutic proteins. Microspheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz andPettit, Bioconjugate Chem. 6:332 (1995); Ranade, “Role of Polymers inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney andBurke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin. Chem.Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres canalso provide carriers for intravenous administration of therapeuticproteins (see, for example, Gref et al., Pharm. Biotechnol. 10:167(1997)).

The present invention also contemplates chemically modified polypeptideshaving binding mouse Zcytor16 activity such as mouse Zcytor16 monomeric,homodimeric, heterodimeric or multimeric soluble receptors, and mouseZcytor16 antagonists, for example anti-mouse Zcytor16 antibodies orbinding polypeptides, which a polypeptide is linked with a polymer, asdiscussed above.

Other dosage forms can be devised by those skilled in the art, as shown,for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5^(th) Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19^(th) Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

As an illustration, pharmaceutical compositions may be supplied as a kitcomprising a container that comprises a polypeptide with a mouseZcytor16 extracellular domain, e.g., mouse Zcytor16 monomeric,homodimeric, heterodimeric or multimeric soluble receptors, or a mouseZcytor16 antagonist (e.g., an antibody or antibody fragment that binds amouse Zcytor16 polypeptide). Therapeutic polypeptides can be provided inthe form of an injectable solution for single or multiple doses, or as asterile powder that will be reconstituted before injection.Alternatively, such a kit can include a dry-powder disperser, liquidaerosol generator, or nebulizer for administration of a therapeuticpolypeptide. Such a kit may further comprise written information onindications and usage of the pharmaceutical composition. Moreover, suchinformation may include a statement that the mouse Zcytor16 compositionis contraindicated in patients with known hypersensitivity to mouseZcytor16.

Polynucleotides and polypeptides of the present invention willadditionally find use as educational tools as a laboratory practicumkits for courses related to genetics and molecular biology, proteinchemistry and antibody production and analysis. Due to its uniquepolynucleotide and polypeptide sequence molecules of mouse Zcytor16 canbe used as standards or as “unknowns” for testing purposes. For example,mouse Zcytor16 polynucleotides can be used as an aid, such as, forexample, to teach a student how to prepare expression constructs forbacterial, viral, and/or mammalian expression, including fusionconstructs, wherein mouse Zcytor16 is the gene to be expressed; fordetermining the restriction endonuclease cleavage sites of thepolynucleotides; determining mRNA and DNA localization of mouse Zcytor16polynucleotides in tissues (i.e., by Northern and Southern blotting aswell as polymerase chain reaction); and for identifying relatedpolynucleotides and polypeptides by nucleic acid hybridization.

Mouse Zcytor16 polypeptides can be used educationally as an aid to teachpreparation of antibodies; identifying proteins by Western blotting;protein purification; determining the weight of expressed mouse Zcytor16polypeptides as a ratio to total protein expressed; identifying peptidecleavage sites; coupling amino and carboxyl terminal tags; amino acidsequence analysis, as well as, but not limited to monitoring biologicalactivities of both the native and tagged protein (i.e., receptorbinding, signal transduction, proliferation, and differentiation) invitro and in vivo. mouse Zcytor16 polypeptides can also be used to teachanalytical skills such as mass spectrometry, circular dichroism todetermine conformation, especially of the four alpha helices, x-raycrystallography to determine the three-dimensional structure in atomicdetail, nuclear magnetic resonance spectroscopy to reveal the structureof proteins in solution. For example, a kit containing the mouseZcytor16 can be given to the student to analyze. Since the amino acidsequence would be known by the professor, the specific protein can begiven to the student as a test to determine the skills or develop theskills of the student, the teacher would then know whether or not thestudent has correctly analyzed the polypeptide. Since every polypeptideis unique, the educational utility of mouse Zcytor16 would be uniqueunto itself.

Moreover, since mouse Zcytor16 has a tissue-specific expression and is apolypeptide with a class II cytokine receptor structure and a distinctexpression pattern, activity can be measured using proliferation assays;luciferase and binding assays described herein. Moreover, expression ofmouse Zcytor16 polynucleotides and polypeptides in lymphoid and othertissues can be analyzed in order to train students in the use ofdiagnostic and tissue-specific identification and methods. Moreovermouse Zcytor16 polynucleotides can be used to train students on the useof chromosomal detection and diagnostic methods. Such assays are wellknown in the art, and can be used in an educational setting to teachstudents about cytokine receptor proteins and examine differentproperties, such as cellular effects on cells, enzyme kinetics, varyingantibody binding affinities, tissue specificity, and the like, betweenmouse Zcytor16 and other cytokine receptor polypeptides in the art.

The antibodies which bind specifically to mouse Zcytor16 can be used asa teaching aid to instruct students how to prepare affinitychromatography columns to purify mouse Zcytor16, cloning and sequencingthe polynucleotide that encodes an antibody and thus as a practicum forteaching a student how to design humanized antibodies. Moreover,antibodies which bind specifically to mouse Zcytor16 can be used as ateaching aid for use in detection e.g., of activated CD91+ cells, cellsorting, or ovarian cancer tissue using histological, and in situmethods amongst others known in the art. The mouse Zcytor16 gene,polypeptide or antibody would then be packaged by reagent companies andsold to universities and other educational entities so that the studentsgain skill in art of molecular biology. Because each gene and protein isunique, each gene and protein creates unique challenges and learningexperiences for students in a lab practicum. Such educational kitscontaining the mouse Zcytor16 gene, polypeptide or antibody areconsidered within the scope of the present invention.

Within one aspect, the present invention provides an isolatedpolypeptide, comprising at least 15 contiguous amino acid residues of anamino acid sequence of SEQ ID NO:38 or SEQ ID NO:48 selected from thegroup consisting of: an amino acid sequence of SEQ ID NO:38 or SEQ IDNO:48 selected from the group consisting of: (a) amino acid residues 24to 230; (b) amino acid residues 27 to 230; (c) amino acid residues 27 to126; and (d) amino acid residues 131 to 230. In one embodiment, theisolated polypeptide disclosed above comprises an amino acid sequence ofSEQ ID NO:38 or SEQ ID NO:48 selected from the group consisting of: (a)amino acid residues 24 to 230; (b) amino acid residues 27 to 230; (c)amino acid residues 27 to 126; and (d) amino acid residues 131 to 230.In another embodiment, the isolated polypeptide disclosed above consistsof an amino acid sequence of SEQ ID NO:38 or SEQ ID NO:48 selected fromthe group consisting of: (a) amino acid residues 24 to 230; (b) aminoacid residues 27 to 230; (c) amino acid residues 27 to 126; and (d)amino acid residues 131 to 230. In another embodiment, the isolatedpolypeptide disclosed above comprises an amino acid sequence that is atleast 90% identical to a reference amino acid sequence of SEQ ID NO:38or SEQ ID NO:48 selected from the group consisting of: (a) amino acidresidues 24 to 230; (b) amino acid residues 27 to 230; (c) amino acidresidues 27 to 126; and (d) amino acid residues 131 to 230. In anotherembodiment, the isolated polypeptide disclosed above comprises an aminoacid sequence of SEQ ID NO:38 or SEQ ID NO:48 selected from the groupconsisting of: (a) amino acid residues 24 to 230; (b) amino acidresidues 27 to 230; (c) amino acid residues 27 to 126; and (d) aminoacid residues 131 to 230.

Within a second aspect, the present invention provides an isolatednucleic acid molecule, wherein the nucleic acid molecule is either (a) anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:39or SEQ ID NO:49, or (b) a nucleic acid molecule that remains hybridizedfollowing stringent wash conditions to a nucleic acid moleculeconsisting of the nucleotide sequence of nucleotides 8 to 697 or 77 to697 or 86 to 697 of SEQ ID NO:37 or SEQ ID NO:47, or the complement ofthe nucleotide sequence of nucleotides 8 to 697 or 77 to 697 or 86 to697 of SEQ ID NO:37 or SEQ ID NO:47. In one embodiment, the isolatednucleic acid molecule is as disclosed above, wherein any differencebetween the amino acid sequence encoded by the nucleic acid molecule andthe corresponding amino acid sequence of SEQ ID NO:38 or SEQ ID NO:48 isdue to a conservative amino acid substitution. In another embodiment,the isolated nucleic acid molecule disclosed above comprises thenucleotide sequence of nucleotides 8 to 697 or 77 to 697 or 86 to 697 ofSEQ ID NO:37 or SEQ ID NO:47.

Within a third aspect, the present invention provides a vector,comprising the isolated nucleic acid molecule as disclosed above.

Within another aspect, the present invention provides an expressionvector, comprising the isolated nucleic acid molecule as disclosedabove, a transcription promoter, and a transcription terminator, whereinthe promoter is operably linked with the nucleic acid molecule, andwherein the nucleic acid molecule is operably linked with thetranscription terminator.

Within another aspect, the present invention provides a recombinant hostcell comprising the expression vector as disclosed above, wherein thehost cell is selected from the group consisting of bacterium, yeastcell, fungal cell, insect cell, mammalian cell, and plant cell.

Within another aspect, the present invention provides a method ofproducing mouse Zcytor16 protein, the method comprising culturingrecombinant host cells that comprise the expression vector as disclosedabove, and that produce the mouse Zcytor16 protein. In one embodiment,the method disclosed above further comprises isolating the mouseZcytor16 protein from the cultured recombinant host cells.

Within another aspect, the present invention provides an antibody orantibody fragment that binds with the polypeptide as disclosed above. Inone embodiment, the antibody disclosed above is selected from the groupconsisting of: (a) polyclonal antibody, (b) murine monoclonal antibody,(c) humanized antibody derived from (b), and (d) human monoclonalantibody.

Within another aspect, the present invention provides an anti-idiotypeantibody that specifically binds with the antibody as disclosed above.

Within another aspect, the present invention provides a fusion protein,comprising the polypeptide as disclosed above. In one embodiment, thefusion protein disclosed above further comprises an immunoglobulinmoiety.

Within another aspect, the present invention provides an isolatedpolynucleotide that encodes a soluble cytokine receptor polypeptidecomprising a sequence of amino acid residues that is at least 90%identical to the amino acid sequence as shown in SEQ ID NO:38 or SEQ IDNO:48 from amino acid 24 to 230, or 27 to 230; and wherein amino aciddifferences consist of conservative amino acid substitutions; andwherein the soluble cytokine receptor polypeptide encoded by thepolynucleotide sequence binds IL-TIF or antagonizes IL-TIF activity. Inone embodiment, the isolated polynucleotide is as disclosed above,wherein the soluble cytokine receptor polypeptide encoded by thepolynucleotide forms a homodimeric, heterodimeric or multimeric receptorcomplex. In another embodiment, the isolated polynucleotide is asdisclosed above, wherein the soluble cytokine receptor polypeptideencoded by the a polynucleotide forms a heterodimeric or multimericreceptor complex further comprising a soluble Class I or Class IIcytokine receptor. In another embodiment, the isolated polynucleotide isas disclosed above, wherein the soluble cytokine receptor polypeptideencoded by the polynucleotide forms a heterodimeric or multimericreceptor complex further comprising a soluble CRF2-4 receptorpolypeptide (SEQ ID NO:35), a soluble IL-10 receptor polypeptide (SEQ IDNO:36), or soluble zcytor11 receptor polypeptide (SEQ ID NO:34).

Within another aspect, the present invention provides an isolatedpolynucleotide that encodes a soluble cytokine receptor polypeptidecomprising a sequence of amino acid residues as shown in SEQ ID NO:38 orSEQ ID NO:48 from amino acid 24 to 230, or 27 to 230, wherein thesoluble cytokine receptor polypeptide encoded by the polynucleotideforms a homodimeric, heterodimeric or multimeric receptor complex. Inone embodiment, the isolated polynucleotide is as disclosed above,wherein the soluble cytokine receptor polypeptide encoded by thepolynucleotide further comprises a soluble Class I or Class II cytokinereceptor. In another embodiment, the isolated polynucleotide is asdisclosed above, wherein the soluble cytokine receptor polypeptideencoded by the polynucleotide forms a heterodimeric or multimericreceptor complex further comprising a soluble CRF2-4 receptorpolypeptide (SEQ ID NO:35), a soluble IL-10 receptor polypeptide (SEQ IDNO:36), or soluble zcytor11 receptor polypeptide (SEQ ID NO:34). Inanother embodiment, the isolated polynucleotide is as disclosed above,wherein the soluble cytokine receptor polypeptide further encodes anintracellular domain. In another embodiment, the isolated polynucleotideis as disclosed above, wherein the soluble cytokine receptor polypeptidefurther comprises an affinity tag.

Within another aspect, the present invention provides an expressionvector comprising the following operably linked elements: (a) atranscription promoter; a first DNA segment encoding a soluble cytokinereceptor polypeptide having an amino acid sequence as shown in SEQ IDNO:38 or SEQ ID NO:48 from amino acid 24 to 230, or 27 to 230; and atranscription terminator; and (b) a second transcription promoter; asecond DNA segment encoding a soluble Class I or Class II cytokinereceptor polypeptide; and a transcription terminator; and wherein thefirst and second DNA segments are contained within a single expressionvector or are contained within independent expression vectors. In oneembodiment, the expression vector disclosed above further comprises asecretory signal sequence operably linked to the first and second DNAsegments. In another embodiment, the expression vector is as disclosedabove, wherein the second DNA segment encodes a polypeptide comprising asoluble CRF2-4 receptor polypeptide (SEQ ID NO:35), a soluble IL-10receptor polypeptide (SEQ ID NO:36), or soluble zcytor11 receptorpolypeptide (SEQ ID NO:34).

Within another aspect, the present invention provides a cultured cellcomprising an expression vector as disclosed above, wherein the cellexpresses the polypeptides encoded by the DNA segments. In oneembodiment, the cultured cell comprising an expression vector is asdisclosed above, wherein the first and second DNA segments are locatedon independent expression vectors and are co-transfected into the cell,and cell expresses the polypeptides encoded by the DNA segments. Inanother embodiment, the cultured cell comprising an expression vector isas disclosed above, wherein the cell expresses a heterodimeric ormultimeric soluble receptor polypeptide encoded by the DNA segments. Inanother embodiment, the cell disclosed above secretes a soluble cytokinereceptor polypeptide heterodimer or multimeric complex. In anotherembodiment, the cell disclosed above secretes a soluble cytokinereceptor polypeptide heterodimer or multimeric complex that binds IL-TIFor antagonizes IL-TIF activity.

Within another aspect, the present invention provides a DNA constructencoding a fusion protein comprising: a first DNA segment encoding apolypeptide having a sequence of amino acid residues as shown in SEQ IDNO:38 or SEQ ID NO:48 from amino acid 24 to 230, or 27 to 230; and atleast one other DNA segment encoding a soluble Class I or Class IIcytokine receptor polypeptide, wherein the first and other DNA segmentsare connected in-frame; and wherein the first and other DNA segmentsencode the fusion protein. In one embodiment, the DNA construct encodinga fusion protein is as disclosed above, wherein at least one other DNAsegment encodes a polypeptide comprising a soluble CRF2-4 receptorpolypeptide (SEQ ID NO:35), a soluble IL-10 receptor polypeptide (SEQ IDNO:36), or soluble zcytor11 receptor polypeptide (SEQ ID NO:34).

Within another aspect, the present invention provides an expressionvector comprising the following operably linked elements: atranscription promoter; a DNA construct encoding a fusion protein asdisclosed above; and a transcription terminator, wherein the promoter isoperably linked to the DNA construct, and the DNA construct is operablylinked to the transcription terminator.

Within another aspect, the present invention provides a cultured cellcomprising an expression vector as disclosed above, wherein the cellexpresses a polypeptide encoded by the DNA construct.

Within another aspect, the present invention provides a method ofproducing a fusion protein comprising: culturing a cell according asdisclosed above; and isolating the polypeptide produced by the cell.

Within another aspect, the present invention provides an isolatedsoluble cytokine receptor polypeptide comprising a sequence of aminoacid residues that is at least 90% identical to an amino acid sequenceas shown in SEQ ID NO:38 or SEQ ID NO:48 from amino acid 24 to 230, or27 to 230, and wherein the soluble cytokine receptor polypeptide bindsIL-TIF or antagonizes IL-TIF activity. In one embodiment, the isolatedpolypeptide is as disclosed above, wherein the soluble cytokine receptorpolypeptide forms a homodimeric, heterodimeric or multimeric receptorcomplex. In another embodiment, the isolated polypeptide is as disclosedabove, wherein the soluble cytokine receptor polypeptide forms aheterodimeric or multimeric receptor complex further comprising asoluble Class I or Class II cytokine receptor. In another embodiment,the isolated polypeptide is as disclosed above, wherein the solublecytokine receptor polypeptide forms a heterodimeric or multimericreceptor complex further comprising a soluble CRF2-4 receptorpolypeptide (SEQ ID NO:35), a soluble IL-10 receptor polypeptide (SEQ IDNO:36), or soluble zcytor11 receptor polypeptide (SEQ ID NO:34).

Within another aspect, the present invention provides an isolatedsoluble cytokine receptor polypeptide comprising a sequence of aminoacid residues as shown in SEQ ID NO:38 or SEQ ID NO:48 from amino acid24 to 230, or 27 to 230, wherein the soluble cytokine receptorpolypeptide forms a homodimeric, heterodimeric or multimeric receptorcomplex. In another embodiment, the isolated polypeptide is as disclosedabove, wherein the solubles cytokine receptor polypeptide forms aheterodimeric or multimeric receptor complex further comprising asoluble Class I or Class II cytokine receptor. In another embodiment,the isolated polypeptide is as disclosed above, wherein the solublecytokine receptor polypeptide forms a heterodimeric or multimericreceptor complex comprising a soluble CRF2-4 receptor polypeptide (SEQID NO:35), a soluble IL-10 receptor polypeptide (SEQ ID NO:36), orsoluble zcytor11 receptor polypeptide (SEQ ID NO:34). In anotherembodiment, the isolated polypeptide is as disclosed above, wherein thesoluble cytokine receptor polypeptide further comprises an affinity tag,chemical moiety, toxin, or label.

Within another aspect, the present invention provides an isolatedheterodimeric or multimeric soluble receptor complex comprising solublereceptor subunits, wherein at least one of the soluble receptor subunitscomprises a soluble cytokine receptor polypeptide comprising a sequenceof amino acid residues as shown in SEQ ID NO:38 or SEQ ID NO:48 fromamino acid 24 to 230, or 27 to 230. In one embodiment, the isolatedheterodimeric or multimeric soluble receptor complex disclosed abovefurther comprises a soluble Class I or Class II cytokine receptorpolypeptide. In another embodiment, the isolated heterodimeric ormultimeric soluble receptor complex disclosed above further comprises asoluble CRF2-4 receptor polypeptide (SEQ ID NO:35), a soluble L-10receptor polypeptide (SEQ ID NO:36), or soluble zcytor11 receptorpolypeptide (SEQ ID NO:34).

Within another aspect, the present invention provides a method ofproducing a soluble cytokine receptor polypeptide that forms aheterodimeric or multimeric complex comprising: culturing a cell asdisclosed above; and isolating the soluble receptor polypeptidesproduced by the cell.

Within another aspect, the present invention provides a method ofproducing an antibody to soluble cytokine receptor polypeptidecomprising: inoculating an animal with a soluble cytokine receptorpolypeptide selected from the group consisting of: (a) a polypeptidecomprising a monomeric or homodimeric soluble cytokine receptorcomprising a polypeptide as shown in SEQ ID NO:38 or SEQ ID NO:48 fromamino acid 24 to 230, or 27 to 230; (b) a polypeptide of (a) furthercomprising a soluble cytokine receptor heterodimeric or multimericreceptor complex comprising a soluble Class I or Class II cytokinereceptor polypeptide; (c) a polypeptide of (a) further comprising asoluble cytokine receptor heterodimeric or multimeric receptor complexcomprising a soluble CRF2-4 receptor polypeptide (SEQ ID NO:35); (d) apolypeptide of (a) further comprising a soluble cytokine receptorheterodimeric or multimeric receptor complex comprising a soluble IL-10receptor polypeptide (SEQ ID NO:36); and wherein the polypeptide elicitsan immune response in the animal to produce the antibody; and isolatingthe antibody from the animal.

Within another aspect, the present invention provides an antibodyproduced by the method as disclosed above, which specifically binds to ahomodimeric, heterodimeric or multimeric receptor complex comprising apolypeptide as shown in SEQ ID NO:38 or SEQ ID NO:48 from amino acid 24to 230, or 27 to 230. In one embodiment, the antibody disclosed above isa monoclonal antibody.

Within another aspect, the present invention provides an antibody whichspecifically binds to a homodimeric, heterodimeric or multimericreceptor complex as disclosed above.

Within another aspect, the present invention provides a method forinhibiting IL-TIF-induced proliferation or differentiation ofhematopoietic cells and hematopoietic cell progenitors comprisingculturing bone marrow or peripheral blood cells with a compositioncomprising an amount of soluble cytokine receptor polypeptide as shownin SEQ ID NO:38 or SEQ ID NO:48 from amino acid 24 to 230, or 27 to 230,sufficient to reduce proliferation or differentiation of thehematopoietic cells in the bone marrow or peripheral blood cells ascompared to bone marrow or peripheral blood cells cultured in theabsence of soluble cytokine receptor. In one embodiment, the method isas disclosed above, wherein the hematopoietic cells and hematopoieticprogenitor cells are lymphoid cells. In another embodiment, the methodis as disclosed above, wherein the lymphoid cells are macrophages or Tcells.

Within another aspect, the present invention provides a method ofreducing IL-TIF-induced or IL-9 induced inflammation comprisingadministering to a mammal with inflammation an amount of a compositionof a polypeptide as shown in SEQ ID NO:38 or SEQ ID NO:48 from aminoacid 24 to 230, or 27 to 230 sufficient to reduce inflammation.

Within another aspect, the present invention, provides a method ofsuppressing an inflammatory response in a mammal with inflammationcomprising: (1) determining a level of serum amyloid A protein; (2)administering a composition comprising a soluble mouse Zcytor16 cytokinereceptor polypeptide as disclosed above in an acceptable pharmaceuticalvehicle; (3) determining a post administration level of serum amyloid Aprotein; (4) comparing the level of serum amyloid A protein in step (1)to the level of serum amyloid A protein in step (3), wherein a lack ofincrease or a decrease in serum amyloid A protein level is indicative ofsuppressing an inflammatory response.

Within another aspect, the present invention provides a method fordetecting a cancer in a patient, comprising: obtaining a tissue orbiological sample from a patient; incubating the tissue or biologicalsample with an antibody as disclosed above under conditions wherein theantibody binds to its complementary polypeptide in the tissue orbiological sample; visualizing the antibody bound in the tissue orbiological sample; and comparing levels of antibody bound in the tissueor biological sample from the patient to a normal control tissue orbiological sample, wherein an increase in the level of antibody bound tothe patient tissue or biological sample relative to the normal controltissue or biological sample is indicative of a cancer in the patient.

Within another aspect, the present invention provides a method fordetecting a cancer in a patient, comprising: obtaining a tissue orbiological sample from a patient; labeling a polynucleotide comprisingat least 14 contiguous nucleotides of SEQ ID NO:37 or SEQ ID NO:47 orthe complement of SEQ ID NO:37 or SEQ ID NO:47; incubating the tissue orbiological sample with under conditions wherein the polynucleotide willhybridize to complementary polynucleotide sequence; visualizing thelabeled polynucleotide in the tissue or biological sample; and comparingthe level of labeled polynucleotide hybridization in the tissue orbiological sample from the patient to a normal control tissue orbiological sample, wherein an increase in the labeled polynucleotidehybridization to the patient tissue or biological sample relative to thenormal control tissue or biological sample is indicative of a cancer inthe patient.

Within another aspect, the present invention provides a transgenicmouse, wherein the mouse over-expresses residues 1 to 230 or 240 to 230,or 27 to 230 of SEQ ID NO:38 or SEQ ID NO:48. In one embodiment, thetransgenic mouse is as disclosed above, wherein the expression ofresidues 1 to 230 or 24 to 230, or 27 to 230 of SEQ ID NO:38 or SEQ IDNO:48 is expressed using a tissue-specific or tissue-restrictedpromoter. In another embodiment, the transgenic mouse is as disclosedabove, wherein the expression of residues 1 to 230 or 24 to 230, or 27to 230 of SEQ ID NO:38 or SEQ ID NO:48 is expressed using anepithelial-specific, colon-specific, or ovary-specific promoter. Inanother embodiment, the transgenic mouse is as disclosed above, whereinthe mouse does not expresses residues 1 to 230 or 24 to 230, or 27 to230 of SEQ ID NO:38 or SEQ ID NO:48, relative to a normal mouse.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Cloning of Mouse Zcytor16 and Construction ofMammalian Expression Vectors that Express Human and Mouse Zcytor16Soluble Receptors: Zcytor16CEE, Zcytor16CFLG, Zcytor16CHIS andZcytor16-Fc4

A. Cloning of Mouse Zcytor16 Extracellular Domain

A mouse ortholog of human Zcytor16 (U.S. patent application Ser. No.09/728,911) was identified and designated “mouse Zcytor16.” Thepolynucleotide sequence of the mouse Zcytor16 clone is shown in SEQ IDNO:37 and SEQ ID NO:47 and polypeptide sequence shown in SEQ ID NO:38and SEQ ID NO:48 respectively.

B. Mammalian Expression Construction of Soluble Zcytor16 receptorZcytor16-Fc4 Construction of Mammalian Expression Vectors that ExpressZcytor16 Soluble Receptor Zcytor16sR/Fc4

An expression vector was prepared to express the soluble human Zcytor16polypeptide. (Zcytor16sR, i.e., from residue 22 (Thr) to residue 231(Pro) of SEQ ID NO:2; SEQ ID NO:13) fused to a C-terminal Fc4 tag (SEQID NO:5).

A PCR generated Zcytor16 DNA fragment of about 630 bp was created usingoligo ZC29,181 (SEQ ID NO:6) and oligo ZC29,182 (SEQ ID NO:7) as PCRprimers to add BamHI and Bgl2 restriction sites at 5′ and 3′ endsrespectively, of the Zcytor16 DNA encoding the soluble receptor. Aplasmid containing the Zcytor16 cDNA (SEQ ID NO:1) (Example 1A) was usedas a template. PCR amplification of the Zcytor16 fragment was performedas follows: One cycle at 94° C. for 1 minute; 25 cycles at 94° C. for 30seconds, 68° C. for 90 seconds, followed by an additional 68° C.incubation for 4 minutes, and hold at 10° C. The reaction was purifiedby chloroform/phenol extraction and isopropanol precipitation, anddigested with BamHI and Bgl2 (Boehringer Mannheim, Indianapolis, Ind.).A band of approximately 630 bp, as visualized by 1% agarose gelelectrophoresis, was excised and the DNA was purified using a QiaexII™purification kit (Qiagen, Valencia, Calif.) according to themanufacturer's instruction.

The Fc4/pzmp20 plasmid is a mammalian expression vector containing anexpression cassette having the CMV promoter, human tPA leader peptide,multiple restriction sites for insertion of coding sequences, a Fc4 tag,and a human growth hormone terminator. The plasmid also has an E. coliorigin of replication, a mammalian selectable marker expression unithaving an SV40 promoter, an enhancer and an origin of replication, aswell as a DHFR gene, and SV40 terminator. The Zcytor16sR/Fc4/pzmp20expression vector uses the human tPA leader peptide (SEQ ID NO:8 and SEQID NO:9) and attaches the Fc4 tag (SEQ ID NO:5) to the C-terminus of theextracellular portion of the Zcytor16 polypeptide sequence. Fc4 is theFc region derived from human IgG, which contains a mutation so that itno longer binds the Fc receptor

About 30 ng of the restriction digested Zcytor16sR insert and about 10ng of the digested vector (which had been cut with Bgl2) were ligated at11° C. overnight. One microliter of ligation reaction was electroporatedinto DH10B competent cells (Gibco BRL, Rockville, Md.) according tomanufacturer's direction and plated onto LB plates containing 50 mg/mlampicillin, and incubated overnight. Colonies were screened byrestriction analysis of DNA which was prepared from 2 ml liquid culturesof individual colonies. The insert sequence of positive clones wasverified by sequence analysis. A large-scale plasmid preparation wasdone using a Qiagen® Mega prep kit (Qiagen) according to manufacturer'sinstruction.

Similar methods are used to prepare mouse Zcytor16-Fc4 fusions, and Fc4fusions of non-Zcytor16 subunits of heterodimeric and multimericreceptors, such as CRF2-4 and IL-10R tagged with Fc4.

C. Construction of Zcytor16 Mammalian Expression Vector containingZcytor16CEE, Zcytor16CFLG and Zcytor16CHIS

An expression vector is prepared for the expression of the soluble,extracellular domain of the Zcytor16 polypeptide (e.g., amino acids22-231 of SEQ ID NO:2; SEQ ID NO:13; or amino acids 24-230 or SEQ IDNO:38 or SEQ ID NO:48), pC4Zcytor16CEE, wherein the construct isdesigned to express a Zcytor16 polypeptide comprised of the predictedinitiating methionine and truncated adjacent to the predictedtransmembrane domain, and with a C-terminal Glu-Glu tag (SEQ ID NO: 10).

A Zcytor16 DNA fragment comprising the Zcytor16 extracellular cytokinebinding domain is created using PCR, and purified. The excised DNA issubcloned into a plasmid expression vector that has a signal peptide,e.g., the native Zcytor16 signal peptide, tPA leader, and attaches aGlu-Glu tag (SEQ ID NO:10) to the C-terminus of the Zcytor16polypeptide-encoding polynucleotide sequence. Such an expression vectormammalian expression vector contains an expression cassette having amammalian promoter, multiple restriction sites for insertion of codingsequences, a stop codon and a mammalian terminator. The plasmid can alsohave an E. coli origin of replication, a mammalian selectable markerexpression unit having an SV40 promoter, enhancer and origin ofreplication, a DHFR gene and the SV40 terminator.

Restriction digested Zcytor16 insert and previously digested vector areligated using standard molecular biological techniques, andelectroporated into competent cells such as DH10B competent cells (GIBCOBRL, Gaithersburg, Md.) according to manufacturer's direction and platedonto LB plates containing 50 mg/ml and incubated overnight. Colonies arescreened by restriction analysis of DNA prepared from individualcolonies. The insert sequence of positive clones is verified by sequenceanalysis. A large-scale plasmid preparation is done using a QIAGEN® Maxiprep kit (Qiagen) according to manufacturer's instructions.

The same process is used to prepare mouse Zcytor16 soluble homodimeric,heterodimeric or multimeric receptors (including non-Zcytor16 solublereceptor subunits, such as, soluble CRF2-4 or IL-10R), or solublereceptors with a C-terminal HIS tag, composed of 6 His residues in a row(SEQ ID NO:12); and a C-terminal FLAG (SEQ ID NO: 11) tag,Zcytor16CFLAG. To construct these constructs, the aforementioned vectorhas either the HIS or the FLAG® tag in place of the glu-glu tag (SEQ IDNO: 10).

Example 2 Transfection And Expression of Soluble Receptor Polypeptides

The day before the transfection, BHK 570 cells (ATCC No. CRL-10314;ATCC, Manasas, Va.) were plated in a 10-cm plate with 50% confluence innormal BHK DMEM (Gibco/BRL High Glucose) media. The day of thetransfection, the cells were washed once with Serum Free (SF) DMEM,followed by transfection with the Zcytor16sR/Fc4/pzmp20 expressionplasmids. Sixteen micrograms of human Zcytor16sR-Fc4 DNA construct(Example 1B) were diluted into a total final volume of 640μl SF DMEM. Adiluted LipofectAMINE™ (Gibco BRL, Gaithersburg, Md.) mixture (35 μlLipofectAMINE in 605 μl SF media) was added to the DNA mix, andincubated for 30 minutes at room temperature. Five milliliters of SFmedia was added to the DNA/LipofectAMINE™ mixture, which was then addedto BHK cells. The cells were incubated at 37° C./5% CO₂ for 5 hours,after which 6.4 ml of BHK media with 10% FBS was added. The cells wereincubated overnight at 37° C./5% CO₂.

Approximately 24 hours post-transfection, the BHK cells were split intoselection media with 1 μM methotrexate (MTX). The cells were repeatedlysplit in this manner until stable Zcytor16sR-Fc4/BHK cell lines wereidentified. To detect the expression level of the Zcytor16 solublereceptor fusion proteins, the transfected BHK cells were washed with PBSand incubated in SF media for 72 hours. The SF condition media wascollected and 20 μl of the sample was run on 10% SDS-PAGE gel underreduced conditions. The protein bands were transferred to nitrocellulosefilter by Western blot, and the fusion proteins were detected usinggoat-anti-human IgG/HRP conjugate (Jackson ImmunoResearch Laboratories,Inc, West Grove, Pa.). An expression vector containing a differentsoluble receptor fused to the Fc4 was used as a control. The expressionlevel of the stable Zcytor16sR-Fc4/BHK cells was approximately 2 mg/L.

For protein purification, the transfected BHK cells were transferredinto T-162 flasks. Once the cells reached about 80% confluence, theywere washed with PBS and incubated in 100 ml SF media for 72 hours, andthen the condition media was collected for protein purification (Example11).

Using the above methods, mouse Zcytor16 soluble receptor fusion proteinsare similarly expressed.

Example 3 Expression of Zcytor16 Soluble Receptor in E. coli

A. Construction of Expression Vector pCZR225 that ExpresseshuZcytor16/MBP-6H Fusion Polypeptide

An expression plasmid containing a polynucleotide encoding a Zcytor16soluble receptor fused C-terminally to maltose binding protein (MBP) isconstructed via homologous recombination. The fusion polypeptidecontains an N-terminal approximately 388 amino acid MBP portion fused tothe human Zcytor16 soluble receptor (e.g., SEQ ID NO:13; or for themouse Zcytor16, amino acids 24 to 230 of SEQ ID NO:38 or SEQ ID NO:48).A fragment of Zcytor16 cDNA (SEQ ID NO:1; or SEQ ID NO:37 or SEQ IDNO:47) is isolated using PCR as described herein. Two primers are usedin the production of the Zcytor16 fragment in a standard PCR reaction:(1) one containing about 40 bp of the vector flanking sequence and about25 bp corresponding to the amino terminus of the Zcytor16, and (2)another containing about 40 bp of the 3′ end corresponding to theflanking vector sequence and about 25 bp corresponding to the carboxylterminus of the Zcytor16. Two μl of the 100 μl PCR reaction is run on a1.0% agarose gel with 1×TBE buffer for analysis, and the expectedapproximately fragment is seen. The remaining PCR reaction is combinedwith the second PCR tube and precipitated with 400 μl of absoluteethanol. The precipitated DNA used for recombining into an appropriatelyrestriction digested recipient vector pTAP98 to produce the constructencoding the MBP-Zcytor16 fusion, as described below.

Plasmid pTAP98 is derived from the plasmids pRS316 and pMAL-c2. Theplasmid pRS316 is a Saccharomyces cerevisiae shuttle vector (Hieter P.and Sikorski, R., Genetics 122:19-27, 1989). pMAL-C2 (NEB) is an E. coliexpression plasmid. It carries the tac promoter driving MalE (geneencoding MBP) followed by a His tag, a thrombin cleavage site, a cloningsite, and the rrnB terminator. The vector pTAP98 is constructed usingyeast homologous recombination. 100 ng of EcoR1 cut pMAL-c2 isrecombined with 1 μg Pvu1 cut pRS316, 1 μg linker, and 1 μg Sca1/EcoR1cut pRS316 are combined in a PCR reaction. PCR products are concentratedvia 100% ethanol precipitation.

Competent yeast cells (S. cerevisiae) are combined with about 10 μl of amixture containing approximately 1 μg of the Zcytor16 receptor PCRproduct above, and 100 ng of digested pTAP98 vector, and electroporatedusing standard methods and plated onto URA-D plates and incubated at 30°C.

After about 48 hours, the Ura+ yeast transformants from a single plateare picked, DNA isolated, and transformed into electrocompetent E. colicells (e.g., MC1061, Casadaban et. al. J. Mol. Biol. 138, 179-207), andplated on MM/CA +AMP 100 mg/L plates (Pryor and Leiting, ProteinExpression and Pruification 10:309-319, 1997) using standard procedures.Cells are grown in MM/CA with 100 μg/ml Ampicillin for two hours,shaking, at 37° C. 1 ml of the culture is induced with 1 mM IPTG. 2-4hours later the 250 μl of each culture is mixed with 250 μl acid washedglass beads and 250 μl Thorner buffer with 5% βME and dye (8M urea, 100mM Tris pH7.0, 10% glycerol, 2 nM EDTA, 5% SDS). Samples are vortexedfor one minute and heated to 65° C. for 10 minutes. 20 μl are loaded perlane on a 4%-12% PAGE gel (NOVEX). Gels are run in 1×MES buffer. Thepositive clones are subjected to sequence analysis.

One microliter of sequencing DNA is used to transform strain BL21. Thecells are electropulsed at 2.0 kV, 25 μF and 400 ohms. Followingelectroporation, 0.6 ml MM/CA with 100 mg/L Ampicillin. Cells are grownin MM/CA and induced with ITPG as described above. The positive clonesare used to grow up for protein purification of the human or mouseZcytor16/MBP-6H fusion protein using standard techniques.

Example 4 Mouse Zcytor16 Soluble Receptor Polyclonal Antibodies

Polyclonal antibodies are prepared by immunizing female New Zealandwhite rabbits with the purified mouse Zcytor16/MBP-6H polypeptide(Example 3), or the purified recombinant mouse Zcytor16CEE or mouseZcytor16-Fc4 soluble receptor (Example 1; Example 11). The rabbits areeach given an initial intraperitoneal (IP) injection of about 200 mg ofpurified protein in Complete Freund's Adjuvant (Pierce, Rockford, Ill.)followed by booster IP injections of 100 mg purified protein inIncomplete Freund's Adjuvant every three weeks. Seven to ten days afterthe administration of the third booster injection, the animals are bledand the serum is collected. The rabbits are then boosted and bled everythree weeks.

The mouse Zcytor16-specific polyclonal antibodies are affinity purifiedfrom the rabbit serum using an CNBr-SEPHAROSE 4B protein column(Pharmacia LKB) that is prepared using about 10 mg of the appropriatepurified mouse Zcytor16 polypeptide per gram CNBr-SEPHAROSE, followed by20× dialysis in PBS overnight. Zcytor16-specific antibodies arecharacterized by an ELISA titer check using 1 mg/ml of the appropriateprotein antigen as an antibody target. The lower limit of detection(LLD) of the rabbit anti-Zcytor16 affinity purified antibodies isdetermined using standard methods.

Example 5 Mouse Zcytor16-Receptor Monoclonal Antibodies

Mouse Zcytor16 receptor Monoclonal antibodies are prepared by immunizingmale BalbC mice (Harlan Sprague Dawley, Indianapolis, Ind.) with thepurified recombinant mouse Zcytor16 proteins described herein. The miceare each given an initial intraperitoneal (IP) injection of 20 mg ofpurified protein in Complete Freund's Adjuvant (Pierce, Rockford, Ill.)followed by booster IP injections of 10 mg purified protein inIncomplete Freund's Adjuvant every two weeks. Seven to ten days afterthe administration of the third booster injection, the animals are bledand the serum is collected, and antibody titer assessed.

Splenocytes are harvested from high-titer mice and fused to murine SP2/0myeloma cells using PEG 1500 (Boerhinger Mannheim, UK) in two separatefusion procedures using a 4:1 fusion ratio of splenocytes to myelomacells (Antibodies: A Laboratory Manual, E. Harlow and D. Lane, ColdSpring Harbor Press). Following 10 days growth post-fusion, specificantibody-producing hybridomas are identified by ELISA using purifiedrecombinant mosue Zcytor16 soluble receptor protein (Example 6C) as anantibody target and by FACS using Baf3 cells expressing the Zcytor16sequence (Example 8) as an antibody target: The resulting hybridomaspositive by both methods are cloned three times by limiting dilution.

Example 6 Assessing Mouse Zcytor16 Receptor Heterodimerization UsingORIGEN Assay

Soluble mouse Zcytor16 receptor (Example 11), or gp130 (Hibi, M. et al.,Cell 63:1149-1157, 1990) are biotinylated by reaction with a five-foldmolar excess of sulfo-NHS-LC-Biotin (Pierce, Inc., Rockford, Ill.)according to the manufacturer's protocol. Soluble mouse Zcytor16receptor and another soluble receptor subunit, for example, solubleEL-10R (sIL-10R) or CRF2-4 receptor (CRF2-4), soluble zcytor11 receptor(U.S. Pat. No. 5,965,704) or soluble zcytor7 receptor (U.S. Pat. No.5,945,511) are labeled with a five fold molar excess of Ru-BPY-NHS(Igen, Inc., Gaithersburg, Md.) according to manufacturer's protocol.The biotinylated and Ru-BPY-NHS-labeled forms of the soluble mouseZcytor16 receptor can be respectively designated Bio-Zcytor16 receptorand Ru-Zcytor16; the biotinylated and Ru-BPY-NHS-labeled forms of theother soluble receptor subunit can be similarly designated. Assays canbe carried out using conditioned media from cells expressing a ligand,such as IL-TIF, that binds mouse Zcytor16 heterodimeric receptors, orusing purified IL-TIF.

For initial receptor binding characterization a panel of cytokines orconditioned medium are tested to determine whether they can mediatehomodimerization of mouse Zcytor16 receptor and if they can mediate theheterodimerization of mouse Zcytor16 receptor with the soluble receptorsubunits described above. To do this, 50 μl of conditioned media orTBS-B containing purified cytokine, is combined with 50 μl of TBS-B (20mM Tris, 150 mM NaCl, 1 mg/ml BSA, pH 7.2) containing e.g., 400 ng/ml ofRu-mZcytor16 receptor and Bio-mZcytor16, or 400 ng/ml of Ru-Zcytor16receptor and e.g., Bio-gp130, or 400 ng/ml of e.g., Ru-CRF2-4 andBio-mZcytor16. Following incubation for one hour at room temperature, 30μg of streptavidin coated, 2.8 mm magnetic beads (Dynal, Inc., Oslo,Norway) are added and the reaction incubated an additional hour at roomtemperature. 200 μl ORIGEN assay buffer (Igen, Inc., Gaithersburg, Md.)is then added and the extent of receptor association measured using anM8 ORIGEN analyzer (Igen, Inc.).

Example 7 Construct for Generating a Mouse Zcytor16 Receptor Heterodimer

A vector expressing a secreted mouse Zcytor16 heterodimer isconstructed. In this construct, the extracellular cytokine-bindingdomain of Zcytor16 (e.g., amino acids 24 to 230 of SEQ ID NO:38 or SEQID NO:48) is fused to the heavy chain of IgG gamma 1 (IgGγ1) while theextracellular portion of the heteromeric cytokine receptor subunit(e.g., an CRF2-4, IL-9, IL-10, zcytor7, zcytor11, IL-4 receptorcomponent) is fused to a human kappa light chain (human κ light chain).

A. Construction of IgG Gamma 1 and Human κ Light Chain Fusion Vectors

The heavy chain of IgGγ1 can be cloned into the Zem229R mammalianexpression vector (ATCC deposit No. 69447) such that any desiredcytokine receptor extracellular domain having a 5′ EcoRI and 3′ NheIsite can be cloned in resulting in an N-terminal extracellulardomain-C-terminal IgGγ1 fusion. The IgGγ1 fragment used in thisconstruct is made by using PCR to isolate the IgGγ1 sequence from aClontech hFetal Liver cDNA library as a template. PCR products arepurified using methods described herein and digested with MluI and EcoRI(Boerhinger-Mannheim), ethanol precipitated and ligated with oligoswhich comprise an desired restriction site linker, into Zem229Rpreviously digested with and EcoRI using standard molecular biologytechniques disclosed herein.

The human κ light chain can be cloned in the Zem228R mammalianexpression vector (ATCC deposit No. 69446) such that any desiredcytokine receptor extracellular domain having a 5′ EcoRI site and a 3′KpnI site can be cloned in resulting in a N-terminal cytokineextracellular domain-C-terminal human κ light chain fusion. As a KpnIsite is located within the human κ light chain sequence, a specialprimer is designed to clone the 3′ end of the desired extracellulardomain of a cytokine receptor into this KpnI site: The primer isdesigned so that the resulting PCR product contains the desired cytokinereceptor extracellular domain with a segment of the human κ light chainup to the KpnI site. This primer preferably comprises a portion of atleast 10 nucleotides of the 3′ end of the desired cytokine receptorextracellular domain fused in frame 5′ to fragment cleaved at the KpnIsite. The human κ light chain fragment used in this construct is made byusing PCR to isolate the human κ light chain sequence from the sameClontech human Fetal Liver cDNA library used above. PCR products arepurified using methods described herein and digested with MluI and EcoRI(Boerhinger-Mannheim), ethanol precipitated and ligated with theMluI/EcoRI linker described above, into Zem228R previously digested withand EcoRI using standard molecular biology techniques disclosed herein.

B. Insertion of Zcytor16 Receptor or Heterodimeric Subunit ExtracellularDomains into Fusion Vector Constructs

Using the construction vectors above, a construct having mouse Zcytor16fused to IgGγ1 is made. This construction is done by PCRing theextracellular cytokine-binding domain of the mouse Zcytor16 receptor(amino acids 24-230 of SEQ ID NO:38 or SEQ ID NO:48) from a placenta orother cDNA library (Clontech) or plasmid (Example 1A) using standardmethods and oligos that provide EcoRI and NheI restriction sites. Theresulting PCR product is digested with EcoRI and NheI, gel purified, asdescribed herein, and ligated into a previously EcoRI and NheI digestedand band-purified Zem229R/IgGγ1 described above. The resulting vector issequenced to confirm that the Zcytor16/IgG gamma 1 fusion is correct.

A separate construct having a heterodimeric cytokine receptor subunitextracellular domain fused to κ light is also constructed as above. TheCRF2-4/human κ light chain construction is performed as above by PCRingfrom, e.g., a lymphocyte cDNA library (Clontech) using standard methods,and oligos that provide EcoRI and KpnI restriction sites. The resultingPCR product is digested with EcoRI and KpnI and then ligating thisproduct into a previously EcoRI and KpnI digested and band-purifiedZem228R/human κ light chain vector described above. The resulting vectoris sequenced to confirm that the cytokine receptor subunit/human κ lightchain fusion is correct.

D. Co-Expression of the Mouse Zcytor16 and Heterodimeric CytokineReceptor Subunit Extracellular Domain

Approximately 15 μg of each of vectors above, are co-transfected intomammalian cells, e.g., BHK-570 cells (ATCC No. CRL-10314) usingLipofectaminePlus™ reagent (Gibco/BRL), as per manufacturer'sinstructions. The transfected cells are selected for 10 days in DMEM+5%FBS (Gibco/BRL) containing 1 μM of methotrexate (MTX) (Sigma, St. Louis,Mo.) and 0.5 mg/ml G418 (Gibco/BRL) for 10 days. The resulting pool oftransfectants is selected again in 10 μm of MTX and 0.5 mg/ml G418 forabout 10 days.

The resulting pool of doubly selected cells is used to generate protein.Three Factories (Nunc, Denmark) of this pool are used to generate 10 Lof serum free conditioned medium. This conditioned media is passed overa 1 ml protein-A column and eluted in about 10, 750 microliterfractions. The fractions having the highest protein concentration arepooled and dialyzed (10 kD MW cutoff) against PBS. Finally the dialyzedmaterial is submitted for amino acid analysis (AAA) using routinemethods.

Example 8 Determination of Receptor Subunits that Heterodimerize orMultimerize with Mouse Zcytor16 Receptor Using a Proliferation Assay

Using standard methods described herein, cells expressing aBaF3/mZcytor16-MPL chimera (wherein the extracellular domain of themouse Zcytor16 (e.g., amino acids 24 to 230 of SEQ ID NO:38 or SEQ IDNO:48) is fused in frame to the intracellular signaling domain of thempl receptor) are tested for proliferative response in the presence ofIL-TIF. Such cells serve as a bioassay cell line to measure ligandbinding of monomeric or homodimeric mouse Zcytor16 receptors. Inaddition, BaF3/mZcytor16-MPL chimera cells transfected with anadditional heterodimeric cytokine receptor subunit can be assessed forproliferative response in the presence of IL-TIF. In the presence ofIL-TIF, if the BaF3/mZcytor16-MPL cells signal, this would suggest thatZcytor16 receptor can homodimerize to signal. Transfection of theBaF3/MPL-mZcytor16 cell line with and additional MPL-class II cytokinereceptor fusion that signals in the presence of the IL-TIF ligand, suchas CRF2-4, determines which heterodimeric cytokine receptor subunits arerequired for mouse Zcytor16 receptor signaling. Use of MPL-receptorfusions for this purpose alleviates the requirement for the presence ofan intracellular signaling domain for the mouse Zcytor16 receptor.

Each independent receptor complex cell line is then assayed in thepresence of IL-TIF and proliferation measured using routine methods(e.g., Alamar Blue assay). The BaF3/MPL-mZcytor16 bioassay cell lineserves as a control for the monomeric or homodimeric receptor activity,and is thus used as a baseline to compare signaling by the variousreceptor complex combinations. The untransfected bioassay cell lineserves as a control for the background activity, and is thus used as abaseline to compare signaling by the various receptor complexcombinations. A BaF3/MPL-mZcytor16 without ligand (IL-TIF) is also usedas a control. The IL-TIF in the presence of the correct receptorcomplex, is expected to increase proliferation of the BaF3/mZcytor16-MPLreceptor cell line approximately 5 fold over background or greater inthe presence of IL-TIF. Cells expressing the components of Zcytor16heterodimeric and multimeric receptors should proliferate in thepresence of IL-TIF.

Example 9 Reconstitution of Mouse Zcytor16 Receptor in Vitro

To identify components involved in the Zcytor16-signaling complex,receptor reconstitution studies are performed as follows. BHK 570 cells(ATCC No. CRL-10314) transfected, using standard methods describedherein, with a luciferase reporter mammalian expression vector plasmidserve as a bioassay cell line to measure signal transduction responsefrom a transfected mouse Zcytor16 receptor complex to the luciferasereporter in the presence of IL-TIF. BHK cells do not endogenouslyexpress the mouse Zcytor16 receptor. An exemplary luciferase reportermammalian expression vector is the KZ134 plasmid that was constructedwith complementary oligonucleotides that contain STAT transcriptionfactor binding elements from 4 genes. A modified c-fos Sis inducibleelement (m67SIE, or hSEE) (Sadowski, H. et al., Science 261:1739-1744,1993), the p21 SIE1 from the p21 WAF1 gene (Chin, Y. et al., Science272:719-722, 1996), the mammary gland response element of the β-caseingene (Schmitt-Ney, M. et al., Mol. Cell. Biol. 11:3745-3755, 1991), anda STAT inducible element of the Fcg RI gene, (Seidel, H. et al., Proc.Natl. Acad. Sci. 92:3041-3045, 1995). These oligonucleotides containAsp718-XhoI compatible ends and are ligated, using standard methods,into a recipient firefly luciferase reporter vector with a c-fospromoter (Poulsen, L. K. et al., J. Biol. Chem. 273:6229-6232, 1998)digested with the same enzymes and containing a neomycin selectablemarker. The KZ134 plasmid is used to stably transfect BHK, or BaF3cells, using standard transfection and selection methods, to make aBHK/KZ134 or BaF3/KZ134 cell line respectively.

The bioassay cell line is transfected with mZcytor16-mpl fusion receptoralone, or co-transfected along with one of a variety of other knownreceptor subunits. Receptor complexes include but are not limited tomZcytor16-mpl receptor only, various combinations of mZcytor16-mplreceptor with one or more of the CRF2-4, IL-9, IL-10, zcytor11, zcytor7class II cytokine receptor subunits, or IL-4 receptor components, or theIL-2 receptor components (IL-2Rα, II-2Rβ, IL-2Rγ); Zcytor16-mpl receptorwith one or more of the IL-4/IL-13 receptor family receptor components(IL-4Rα, IL13Rα, IL-13Rα′), as well as other Interleukin receptors(e.g., IL-15 Rα; IL-7Rα, IL-9Rα, IL-21R (Zcytor16)). Each independentreceptor complex cell line is then assayed in the presence ofcytokine-conditioned media or purified cytokines and luciferase activitymeasured using routine methods. The untransfected bioassay cell lineserves as a control for the background luciferase activity, and is thusused as a baseline to compare signaling by the various receptor complexcombinations. The conditioned medium or cytokine that binds themousezyctor16 receptor in the presence of the correct receptor complex,is expected to give a luciferase readout of approximately 5 fold overbackground or greater.

As an alternative, a similar assay can be performed whereinBaf3/mZcytor16-mpl cell lines are co-transfected as described above andproliferation measured (Example 8).

Example 10 COS Cell Transfection and Secretion Trap

COS cell transfections were performed as follows: A mixture of 0.5 μgDNA and 5 μl lipofectamine (Gibco BRL) in 92 ul serum free DMEM media(55 mg sodium pyruvate, 146 mg L-glutamine, 5 mg transferrin, 2.5 mginsulin, 1 μg selenium and 5 mg fetuin in 500 ml DMEM) was incubated atroom temperature for 30 minutes and then 400 μl serum free DMEM mediaadded. A 500 μl mixture was added onto COS cells plated on 12-welltissue culture plate at 1.5×10⁵ COS cells/well and previously incubatedfor 5 hours at 37° C. An additional 500 μl 20% FBS DMEM media (100 mlFBS, 55 mg sodium pyruvate and 146 mg L-glutamine in 500 ml DMEM) wasadded and the plates were incubated overnight.

The secretion trap was performed as follows: Media was rinsed off cellswith PBS and fixed for 15 minutes with 1.8% Formaldehyde in PBS. Cellswere then washed with TNT (0.1M Tris-HCl, 0.15M NaCl, and 0.05% Tween-20in H₂O). Cells were permeated with 0.1% Triton-X in PBS for 15 minutesand washed again with TNT. Cells were blocked for 1 hour with TNB (0.1MTris-HCl, 0.15M NaCl and 0.5% Blocking Reagent (NEN RenaissanceTSA-Direct Kit, NEN) in H₂O. Cells were again washed with TNT. Cellswere then incubated for 1 hour with 1-3 μg/ml human Zcytor16 solublereceptor Fc4 fusion protein (Zcytor16sR-Fc4) (Example 11) in TNB. Cellswere washed with TNT, and then incubated for another hour with 1:200diluted goat-anti-human Ig-HRP (Fc specific; Jackson ImmunoResearchLaboratories, Inc.) in TNB. Cells were again washed with TNT. Antibodiespositively binding to the human Zcytor16sR-Fc4 were detected withfluorescein tyramide reagent diluted 1:50 in dilution buffer (NEN kit)and incubated for 4-6 minutes. Cells were again washed with TNT. Cellswere preserved with Vectashield Mounting Media (Vector Labs) diluted 1:5in TNT. Cells were visualized using FITC filter on fluorescentmicroscope.

Since Zcytor16 is a Class II cytokine receptor, the binding of humanZcytor16sR/Fc4 fusion protein with known or orphan Class II cytokineswas tested. The pZP7 expression vectors containing cDNAs of cytokines(including IL-TIF, interferon alpha, interferon beta, interferon gamma,IL-10, amongst others were transfected into COS cells, and the bindingof Zcytor16sR/Fc4 to transfected COS cells were carried out using thesecretion trap assay described above. Human IL-TIF showed positivebinding. Based on these data, human IL-TIF and human Zcytor16 is apotential ligand-receptor pair. Similar methods are used to show thatmouse Zcytor16 is an orthologous class II receptor that binds IL-TIF.

Example 11 Purification of Zcytor16-Fc4 Polypeptide from Transfected BHK570 Cells

Unless otherwise noted, all operations were carried out at 4° C. Thefollowing procedure was used for purifying human Zcytor16 polypeptidecontaining C-terminal fusion to human Fc4 (Zcytor16-Fc4; Example 1).About 16,500 ml of conditioned media from BHK 570 cells transfected withhuman Zcytor16-Fc4 (Example 2) was filtered through a 0.2 um sterilizingfilter and then supplemented with a solution of protease inhibitors, tofinal concentrations of, 0.001 mM leupeptin (Boerhinger-Mannheim,Indianapolis, Ind.), 0.001 mM pepstatin (Boerhinger-Mannheim) and 0.4 mMPefabloc (Boerhinger;-Mannheim). A Poros protein A50 column (20 ml bedvolume, Applied Biosystems) was packed and washed with 400 ml PBS(Gibco/BRL) The supplemented conditioned media was passed over thecolumn with a flow rate of 15 ml/minute, followed by washing with 800 mlPBS (BRL/Gibco). Zcytor16-Fc4 was eluted from the column with 0.1 MGlycine pH 3.0 and 5 ml fractions were collected directly into 0.5 ml 2MTris pH 7.8, to adjust the final pH to 7.4 in the fractions.

Column performance was characterized through western blotting ofreducing SDS-PAGE gels of the starting media and column pass through.Western blotting used anti-human IgG HRP (Amersham) antibody, whichshowed an immunoreactive protein at 60,000 Da in the starting media,with nothing in the pass through, suggesting complete capture. Theprotein A50 eluted fractions were characterized by reducing SDS PAGEgel. This gel showed an intensely Coomassie stained band at 60,000 Da infractions 3 to 11. Fractions 3 to 11 were pooled.

Protein A 50 elution pool was concentrated from 44 ml to 4 ml using a30,000 Da Ultrafree Biomax centrifugal concentrator (15 ml volume,Millipore). A Sephacryl S-300 gel filtration column (175 ml bed volume;Pharmacia) was washed with 350 ml PBS (BRL/Gibco). The concentrated poolwas injected over the column with a flow rate of 1.5 ml/min, followed bywashing with 225 ml PBS (BRL/Gibco). Eluted peaks were collected into 2ml fractions.

Eluted fractions were characterized by reducing and non-reducing silverstained (Geno Technology) SDS PAGE gels. Reducing silver stained SDSPAGE gels showed an intensely stained band at 60,000 Da in fractions14-31, while non-reducing silver stained SDS PAGE gels showed anintensely stained band at 160,000 Da in fractions 14-31. Fractions 1-13showed many bands of various sizes. Fractions 14-31 were pooled,concentrated to 22 ml using 30,000 Da Ultrafree Biomax centrifugalconcentrator (15 ml volume, Millipore). This concentrate was filteredthrough a 0.2 μm Acrodisc sterilizing filter (Pall Corporation).

The protein concentration of the concentrated pooled fractions wasperformed by BCA analysis (Pierce, Rockford, Ill.) and the material wasaliquoted, and stored at −80° C. according to our standard procedures.The concentration of the pooled fractions was 1.50 mg/ml.

This method is also used to purify the mouse Zcytor16-Fc4 andheterologous class II receptor Fc4 fusions.

Example 12 Human and Mouse Zcytor16 Tissue Distribution in Tissue PanelsUsing Northern Blot and PCR

A. Human Zcytor16 Tissue Distribution using Northern Blot and Dot Blot

Northern blot analysis was performed using Human Multiple TissueNorthern Blots I, II, III (Clontech) and an in house generated U-937northern blot. U-937 is a human monoblastic promonocytic cell line. ThecDNA probe was generated using oligos ZC25,963 (SEQ ID NO: 16) andZC28,354 (SEQ ID NO: 17). The PCR conditions were as follows: 94° for 1minute; 30 cycles of 94°, 15 seconds; 60°, 30 seconds; 72°, 30 secondsand a final extension for 5 minutes at 72°. The 364 bp product was gelpurified by gel electrophoresis on a 1% TBE gel and the band was excisedwith a razor blade. The cDNA was extracted from the agarose using theQIAquick Gel Extraction Kit (Qiagen). 94 ng of this fragment wasradioactively labeled with ³²P-dCTP using Rediprime II (Amersham), arandom prime labeling system, according to the manufacturer'sspecifications. Unincorporated radioactivity was removed using aNuc-Trap column (Stratagene) according to manufacturer's instructions.Blots were prehybridized at 65° for 3 hours in ExpressHyb (Clontech)solution. Blots were hybridized overnight at 65° in Expresshyb solutioncontaining 1.0×10⁶ cpm/ml of labeled probe, 0.1 mg/ml of salmon spermDNA and 0.5 μg/ml of human cot-1 DNA. Blots were washed in 2×SSC, 0.1%SDS at room temperature with several solution changes then washed in0.1×SSC. 0.1% SDS at 55° for 30 minutes twice. Transcripts ofapproximately 1.6 kb and 3.0 kb size were detected in spleen andplacenta, but not other tissues examined. The same sized transcriptsplus an additional approximate 1.2 kb transcript was detected in U-937cell line.

B. Tissue Distribution of human Zcytor16 in Tissue cDNA Panels Using PCR

A panel of cDNAs from human tissues was screened for Zcytor16 expressionusing PCR. The panel was made in-house and contained 77 marathon cDNAand cDNA samples from various normal and cancerous human tissues andcell lines are shown in Table 5, below. The cDNAs came from in-houselibraries or marathon cDNAs from in-house RNA preps, Clontech RNA, orInvitrogen RNA. The marathon cDNAs were made using the marathon-Ready™kit (Clontech, Palo Alto, Calif.) and QC tested with clathrin primersZC21195 (SEQ ID NO:18) and ZC21196 (SEQ ID NO:19) and then diluted basedon the intensity of the clathrin band. To assure quality of the panelsamples, three tests for quality control (QC) were run: (1) To assessthe RNA quality used for the libraries, the in-house cDNAs were testedfor average insert size by PCR with vector oligos that were specific forthe vector sequences for an individual cDNA library; (2) Standardizationof the concentration of the cDNA in panel samples was achieved usingstandard PCR methods to amplify full length alpha tubulin or G3PDH cDNAusing a 5′ vector oligo ZC14,063 (SEQ ID NO:20) and 3′ alpha tubulinspecific oligo primer ZC17,574 (SEQ ID NO:21) or 3′ G3PDH specific oligoprimer ZC17,600 (SEQ ID NO:22); and (3) a sample was sent to sequencingto check for possible ribosomal or mitochondrial DNA contamination. Thepanel was set up in a 96-well format that included a human genomic DNA(Clontech, Palo Alto, Calif.) positive control sample. Each wellcontained approximately 0.2-100 pg/μl of cDNA.

The PCR reactions were set up using oligos ZC25,963 (SEQ ID NO:30) andZC25,964 (SEQ ID NO:31), Advantage 2 DNA Polymerase Mix (Clontech) andRediload dye (Research Genetics, Inc., Huntsville, Ala.). Theamplification was carried out as follow: 1 cycle at 94° C. for 1 minute,38 cycles of 94° C. for 10 seconds, 60° C. for 30 seconds and 72° C. for30 seconds, followed by 1 cycle at 72° C. for 5 minutes. The correctpredicted DNA fragment size was observed in bone marrow, fetal heart,fetal kidney, fetal muscle, fetal skin, heart, mammary gland, placenta,salivary gland, skeletal muscle, small intestine, spinal cord, spleen,kidney, fetal brain, esophageal tumor, uterine tumor, stomach tumor,ovarian tumor, rectal tumor, lung tumor and RPMI-1788 (a B-lymphocytecell line). Zcytor16 expression was not observed in the other tissuesand cell lines tested in this panel. The expression pattern of Zcytor16shows expression in certain tissue-specific tumors especially, e.g.,ovarian cancer, stomach cancer, uterine cancer, rectal cancer, lungcancer and esophageal cancer, where zcytor 16 is not expressed in normaltissue, but is expressed in the tumor tissue. One of skill in the artwould recognize that the polynucleotides, polypeptides, antibodies, andbinding partners of the present invention can be used as a diagnostic todetect cancer, or cancer tissue in a biopsy, tissue, or histologicsample, particularly e.g., ovarian cancer, stomach cancer, uterinecancer, rectal cancer, lung cancer and esophageal cancer tissue. Suchdiagnostic uses for the molecules of the present invention are known inthe art and described herein.

In addition, because the expression pattern of human Zcytor16, one ofIL-TIF's receptors, shows expression in certain specific tissues as wellas tissue-specific tumors, binding partners including the naturalliganed, IL-TIF, can also be used as a diagnostic to detect specifictissues (normal or abnormal), cancer, or cancer tissue in a biopsy,tissue, or histologic sample, where IL-TIF receptors are expressed, andparticularly e.g., ovarian cancer, stomach cancer, uterine cancer,rectal cancer, lung cancer and esophageal cancer tissue. IL-TIF can alsobe used to target other tissues wherein its receptors, e.g., Zcytor16and zcytor11 are expressed. Moreover, such binding partners could beconjugated to chemotherapeutic agents, toxic moieties and the like totarget therapy to the site of a tumor or diseased tissue. Suchdiagnostic and targeted therapy uses are known in the art and describedherein.

A commercial 1st strand cDNA panel (Human Blood Fractions MTC Panel,Clontech, Palo Alto, Calif.) was also assayed as above. The panelcontained the following samples: mononuclear cells, activatedmononuclear cells, resting CD4+ cells, activated CD4+ cells, restingCD8+ cells, activated CD8+cells, resting CD14+ cells, resting CD19+cells and activated CD19+ cells. Activated CD4+ cells and activatedCD19+ cells showed Zcytor16 expression, whereas the other cells tested,including resting CD4+ cells and resting CD19+ cells, did not. TABLE 5Tissue #samples adrenal gland 1 bone marrow 3 cervix 1 fetal brain 3fetal kidney 1 fetal lung 1 heart 2 kidney 2 lung 1 mammary gland 1ovary 1 pituitary 2 prostate 3 salivary gland 2 small intestine 1 spleen1 stomach 1 testis 5 thymus 1 thyroid 2 trachea 1 esophageal tumor 1liver tumor 1 rectal tumor 1 uterine tumor 2 HaCAT library 1 HPVSlibrary 1 K562 1 bladder 1 brain 2 colon 1 fetal heart 2 fetal liver 2fetal skin 1 fetal muscle 1 liver 1 lymph node 1 melanoma 1 pancreas 1placenta 3 rectum 1 skeletal muscle 1 spinal cord 2 uterus 1 adipocytelibrary 1 islet 1 prostate SMC 1 RPMI 1788 1 WI38 1 lung tumor 1 ovariantumor 1 stomach tumor 1 CD3+ library 1 HPV library 1 MG63 library 1C. Tissue Distribution of Human Zcytor16 in Human Tissue and Cell LineRNA Panels Using RT-PCR

A panel of RNAs from human cell lines was screened for human Zcytor16expression using RT-PCR. The panels were made in house and contained 84RNAs from various normal and cancerous human tissues and cell lines asshown in Tables 6-9 below. The RNAs were made from in house or purchasedtissues and cell lines using the RNAeasy Midi or Mini Kit (Qiagen,Valencia, Calif.). The panel was set up in a 96-well format with 100 ngsof RNA per sample. The RT-PCR reactions were set up using oligosZC25,963 (SEQ ID NO:30) and ZC25,964 (SEQ ID NO:31), Rediload dye andSUPERSCRIPT One Step RT-PCR System (Life Technologies, Gaithersburg,Md.). The amplification was carried out as follows: one cycle at 55° for30 minutes followed by 40 cycles of 94°, 15 seconds; 59°, 30 seconds;72°, 30 seconds; then ended with a final extension at 72° for 5 minutes.8 to 10 μls of the PCR reaction product was subjected to standardAgarose gel electrophoresis using a 4% agarose gel. The correctpredicted cDNA fragment size of 184 bps was observed in cell linesU-937, HL-60, ARPE-19, HaCat#1, HaCat#2, HaCat#3, and HaCat#4; bladder,cancerous breast, normal breast adjacent to a cancer, bronchus, colon,ulcerative colitis colon, duodenum, endometrium, esophagus,gastro-esophageal, heart left ventricle, heart ventricle, ileum, kidney,lung, lymph node, lymphoma, mammary adenoma, mammary gland, cancerousovary, pancreas, parotid and skin, spleen lymphoma and small bowel.Zcytor16 expression was not observed in the other tissues and cell linestested in this panel.

Zcytor16 is detectably expressed by PCR in normal tissues: such as, thedigestive system, e.g., esophagus, gastro-esophageal, pancreas,duodenum, lleum, colon, small bowel; the female reproductive system,e.g., mammary gland, endometrium, breast (adjacent to canceroustissues); and others systems, e.g., lymph nodes, skin, parotid, bladder,bronchus, heart ventricles, and kidney. Moreover, Zcytor16 is detectablyexpressed by PCR in several human tumors: such as tumors associated withfemale reproductive tissues e.g., mammary adenoma, ovary cancer, uterinecancer, other breast cancers; and other tissues such as lymphoma,stomach tumor, and lung tumor. The expression of Zcytor16 is found innormal tissues of female reproductive organs, and in some tumorsassociated with these organs. As such, Zcytor16 can serve as a markerfor these tumors wherein the Zcytor16 may be over-expressed. Severalcancers positive for Zcytor16 are associated with ectodermal/epithelialorigin (mammary adenoma, and other breast cancers). Hence, Zcytor16 canserve as a marker for epithelial tissue, such as epithelial tissues inthe digestive system and female reproductive organs (e.g., endometrialtissue, columnar epithelium), as well as cancers involving epithelialtissues. Moreover, in a preferred embodiment, Zcytor16 can serve as amarker for certain tissue-specific tumors especially, e.g., ovariancancer, stomach cancer, uterine cancer, rectal cancer, lung cancer andesophageal cancer, where zcytor 16 is not expressed in normal tissue,but is expressed in the tumor tissue. Use of polynucleotides,polypeptides, and antibodies of the present invention for diagnosticpurposes are known in the art, and disclosed herein. TABLE 6 Tissue#samples adrenal gland 6 bladder 3 brain 2 brain meningioma 1 breast 1cancerous breast 4 normal breast adjacent to cancer 5 bronchus 3 colon15 cancerous colon 1 normal colon adjacent to cancer 1 ulcerativecolitis colon 1 duodenum 1 endometrium 5 cancerous endometrium 1 gastriccancer 1 esophagus 7 gastro-esophageal 1 heart aorta 1 heart leftventricle 4 heart right ventricle 2 heart ventricle 1 ileum 3 kidney 15cancerous kidney 1

TABLE 7 Tissue/Cell Line #samples 293 1 C32 1 HaCat#1 1 HaCat#2 1HaCat#3 1 HaCat#4 1 WI-38 1 WI-38 + 2 um ionomycin#1 1 WI-38 + 2 umionomycin#2 1 WI-38 + 5 um ionomycin#1 1 WI-38 + 5 um ionomycin#2 1Caco-2, 1 Caco-2, differentiated 1 DLD-1 1 HRE 1 HRCE 1 MCF7 1 PC-3 1TF-1 1 5637 1 143B 1 ME-180 1 prostate epithelia 1 U-2 OS 1 T-47D 1Mg-63 1 Raji 1 U-373 MG 1 A-172 1 CRL-1964 1 CRL-1964 + butryic acid 1HUVEC 1 SK-Hep-1 1 SK-Lu-1 1 Sk-MEL-2 1 K562 1 BeWo 1 FHS74.Int 1 HL-601 Malme 3M 1 FHC 1 HREC 1 HBL-100 1 Hs-294T 1 Molt4 1 RPMI 1 U-937 1A-375 1 HCT-15 1 HT-29 1 MRC-5 1 RPT-1 1 RPT-2 1 WM-115 1 A-431 1WERI-Rb-1 1 HEL-92.1.7 1 HuH-7 1 MV-4-11 1 U-138 1 CCRF-CEM 1 Y-79 1A-549 1 EL-4 1 HeLa 229 1 HUT 78 1 NCI-H69 1 SaOS2 1 USMC 1 UASMC 2AoSMC 1 UtSMC 1 HepG2 1 HepG2-IL6 1 NHEK#1 1 NHEK#2 1 NHEK#3 1 NHEK#4 1ARPE-19 1 G-361 1 HISM 1 3AsubE 1 INT407 1

TABLE 8 Tissue #samples liver 10 lymph node 1 lymphoma 4 mammary adenoma1 mammary gland 3 melinorioma 1 osteogenic sarcoma 2 pancreas 4 skin 5sarcoma 2 lung 13 cancerous lung 2 normal lung adjacent to cancer 1muscle 3 neuroblastoma 1 omentum 2 ovary 6 cancerous ovary 2 parotid 7salivary gland 4

TABLE 9 Tissue #samples small bowel 10 spleen 3 spleen lymphoma 1stomach 13 stomach cancer 1 uterus 11 uterine cancer 1 thyroid 9D. Tissue Distribution of Mouse Zcytor16 in Tissue Panels Using PCR

A panel of cDNAs from murine tissues was screened for mouse Zcytor16expression using PCR. The panel was made in-house and contained 49marathon cDNA and cDNA samples from various normal and cancerous murinetissues and cell lines are shown in Table 10, below. The cDNAs areeither in-house cDNA libraries marathon cDNAs. The RNA used to createthese cDNA's came from either in-house RNA preps, Clontech RNA, orInvitrogen RNA. The mouse marathon cDNAs were made using themarathon-Ready™ kit (Clontech, Palo Alto, Calif.) and QC tested withmouse transferrin receptor primers ZC10,651 (SEQ ID NO:42) and ZC10,565(SEQ ID NO:43) and then diluted based on the intensity of thetransferrin band. The in-house libraries were diluted to provide 25 ngper well of cDNA. To assure quality of the amplified library samples inthe panel, three tests for quality control (QC) were run: (1) To assessthe RNA quality used for the libraries, the in-house cDNAs were testedfor average insert size by PCR with vector oligos that were specific forthe vector sequences for an individual cDNA library; (2) Standardizationof the concentration of the cDNA in panel samples was achieved usingstandard PCR methods to amplify full length alpha tubulin or G3PDH cDNAusing a 5′ vector oligo: ZC14,063 (SEQ ID NO:20) and 3′ alpha tubulinspecific oligo primer ZC17,574 (SEQ ID NO:21) or 3′ G3PDH specific oligoprimer ZC17,600 (SEQ ID NO:22); and (3) a sample was sent to sequencingto check for possible ribosomal or mitochondrial DNA contamination. Thepanel was set up in a 96-well format that included a mouse genomic DNA(Clontech, Palo Alto, Calif.) positive control sample. Each wellcontained approximately 0.2-100 pg/μl of cDNA. The PCR was set up usingoligos ZC38,001 (SEQ ID NO:44) and ZC38,022 (SEQ ID NO:45), Advantage 2Taq Polymerase (Clontech, Palo Alto, Calif.), and Rediload dye (ResearchGenetics, Inc., Huntsville, Ala.). The amplification was carried out asfollows: 1 cycle at 94° C. for 2 minutes; 35 cycles of 94° C. for 10seconds, 60° C. for 20 seconds and 68° C. for 30 seconds, followed by 1cycle at 68° C. for 7 minutes. About 12.5 μl of the PCR reaction productwas subjected to standard Agarose gel electrophoresis using a ˜4%agarose gel.

The correct predicted DNA fragment size of 114 base pairs was observedin Lung, Pancreas, Placenta, Salivary Gland, Skeletal, Muscle, Skin,Small, Intestine, Smooth Muscle, Spleen, Stomach, and Testis cDNAs.These initial expression results for the mouse Zcytor16 corroborated theexpression data seen for human Zcytor16 discussed herein, suggestingthat the use of mouse Zcytor16 in an animal model, such as mouse, wouldreasonably reflect the in vivo expression and function of Zcytor16 seenin humans, and discussed herein. TABLE 7 Tissue/Cell line #samples 229 17F2 1 Adipocytes-Amplified 1 aTC1.9 1 Brain 4 CCC4 1 CD90+ Amplified 1OC10B 1 Dentritic 1 Embyro 1 Heart 2 Kidney 3 Liver 2 Lung 2 MEWt#2 1P388D1 1 Pancreas 1 Placenta 2 Jakotay-Prostate Cell Line 1Nelix-Prostate Cell Line 1 Paris-Prostate Cell Line 1 Torres-ProstateCell Line 1 Tuvak-Prostate Cell Line 1 Salivary Gland 2 Skeletal Muscle1 Skin 2 Small Intestine 1 Smooth Muscle 2 Spleen 2 Stomach 1 Testis 3Thymus 1

Example 13 Construction of Expression Vector Expressing Full-LengthZcytor11

The entire zcytor11 receptor (commonly owned U.S. Pat. No. 5,965,704)was isolated by digestion with EcoRI and XhoI from plasmid pZP7P,containing full-length zcytor11 receptor cDNA (SEQ ID NO:24; amino acidsequence in SEQ ID NO:25) and a puromycin resistance gene. The digestwas run on a 1% low melting point agarose (Boerhinger Mannheim) gel andthe approximately 1.5 kb zcytor11 cDNA was isolated using Qiaquick™ gelextraction kit (Qiagen) as per manufacturer's instructions. The purifiedzcytor11 cDNA was inserted into an expression, vector as describedbelow.

Recipient expression vector pZP7Z was digested with EcoRI (BRL) and XhoI(Boehringer Mannheim) as per manufacturer's instructions, and gelpurified as described above. This vector fragment was combined with theEcoRI and XhoI cleaved zcytor11 fragment isolated above in a ligationreaction. The ligation was run using T4 Ligase (BRL) at 12° C.overnight. A sample of the ligation was electroporated in to DH10BelectroMAX™ electrocompetent E. coli cells (25 μF, 200Ω, 1.8V).Transformants were plated on LB+Ampicillin plates, and single colonieswere picked into 2 ml LB+Ampicillin and grown overnight. Plasmid DNA wasisolated using Wizard Minipreps (Promega), and each was digested withEcoRI and XhoI to confirm the presence of insert. The insert wasapproximately 1.5 kb, and was full-length. Digestion with SpeI and PstIwas used to confirm the identity of the vector.

Example 14 Construction of BaF3 Cells Expressing the CRF2-4 Receptor(BaF3/CRF2-4 Cells) and BaF3 Cells Expressing the CRF2-4 Receptor withthe Zcytor11 Receptor (BaF3/CRF2-4/zcytor11 Cells)

BaF3 cells expressing the full-length CFR2-4 receptor were constructed,using 30 μg of a CFR2-4 expression vector, described below. The BaF3cells expressing the CFR2-4 receptor were designated as BaF3/CFR2-4.These cells were used as a control, and were further transfected withfull-length zcytor11 receptor (U.S. Pat. No. 5,965,704) and used toconstruct a screen for IL-TIF activity as described below.

A. Construction of BaF3 Cells Expressing the CRF2-4 Receptor

The full-length cDNA sequence of CRF2-4 (Genbank Accession No. Z17227)was isolated from a Daudi cell line cDNA library, and then cloned intoan expression vector pZP7P.

BaF3, an interleukin-3 (IL-3 ) dependent pre-lymphoid cell line derived,from murine bone marrow (Palacios and Steinmetz, Cell 41: 727-734, 1985;Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986), wasmaintained in complete media (RPMI medium (JRH Bioscience Inc., Lenexa,Kans.) supplemented with 10% heat-inactivated fetal calf serum, 2 ng/mlmurine IL-3 (mIL-3) (R & D, Minneapolis, Minn.), 2 mM L-glutaMax-1™(Gibco BRL), 1 mM Sodium Pyruvate (Gibco BRL), and PSN antibiotics(GIBCO BRL)). Prior to electroporation, CRF2-4/pZP7P was prepared andpurified using a Qiagen Maxi Prep kit (Qiagen) as per manufacturer'sinstructions. For electroporation, BaF3 cells were washed once inserum-free RPMI media and then resuspended in serum-free RPMI media at acell density of 10⁷ cells/ml. One ml of resuspended BaF3 cells was mixedwith 30 μg of the CRF2-4/pZP7P plasmid DNA and transferred to separatedisposable electroporation chambers (GIBCO BRL). Following a 15-minuteincubation at room temperature the cells were given two serial shocks(800 IFad/300 V.; 1180 IFad/300 V.) delivered by an electroporationapparatus (CELL-PORATOR™; GIBCO BRL). After a 5-minute recovery time,the electroporated cells were transferred to 50 ml of complete media andplaced in an incubator for 15-24 hours (37° C., 5% CO₂). The cells werethen spun down and resuspended in 50 ml of complete media containing 2μg/ml puromycin in a T-162 flask to isolate the puromycin-resistantpool. Pools of the transfected BaF3 cells, hereinafter calledBaF3/CRF2-4 cells, were assayed for signaling capability as describedbelow. Moreover these cells were further transfected with zcytor11receptor as described below.

B. Construction of BaF3 Cells Expressing CRF2-4 and Zcytor11 Receptors

BaF3/CRF2-4 cells expressing the full-length zcytor11 receptor wereconstructed as per Example 5A above, using 30 μg of the zcytor11expression vector, described in Example 6 above. Following recovery,transfectants were selected using 200 μg/ml zeocin and 2 μg/mlpuromycin. The BaF3/CRF2-4 cells expressing the zcytor11 receptor weredesignated as BaF3/CRF2-4/zcytor11 cells. These cells were used toscreen for IL-TIF activity as well as Zcytor16 antagonist activitydescribed in Example 15.

Example 15 Screening for IL-TIF Antagonist Activity UsingBaF3/CRF2-4/zcytor11 Cells Using an Alamar Blue Proliferation Assay

A. Screening for IL-TIF Activity Using BaF3/CRF2-4/zcytor11 Cells Usingan Alamar Blue Proliferation Assay

Purified human IL-TIF-CEE (Example 19) was used to test for the presenceof proliferation activity as described below. Purified humsnZcytor16-Fc4 (Example 11) was used to antagonize the proliferativeresponse of the IL-TIF in this assay as described below.

BaF3/CRF2-4/zcytor11 cells were spun down and washed in the completemedia, described in Example 7A above, but without mIL-3 (hereinafterreferred to as “mIL-3 free media”). The cells were spun and washed 3times to ensure the removal of the mIL-3. Cells were then counted in ahemacytometer. Cells were plated in a 96-well format at 5000 cells perwell in a volume of 100 μl per well using the mIL-3 free media.

Proliferation of the BaF3/CRF2-4/zcytor11 cells was assessed usingIL-TIF-CEE protein diluted with mIL-3 free media to 50, 10, 2, 1, 0.5,0.25, 0.13, 0.06 ng/ml concentrations. 100 μl of the diluted protein wasadded to the BaF3/CRF2-4/zcytor11 cells. The total assay volume is 200μl. The assay plates were incubated at 37° C., 5% CO₂ for 3 days atwhich time Alamar Blue (Accumed, Chicago, Ill.) was added at 20 μl/well.Plates were again incubated at 37° C., 5% CO₂ for 24 hours. Alamar Bluegives a fluourometric readout based on number of live cells, and is thusa direct measurement of cell proliferation in comparison to a negativecontrol. Plates were again incubated at 37° C., 5% CO₂ for 24 hours.Plates were read on the Fmax™ plate reader (Molecular Devices Sunnyvale,Calif.) using the SoftMax™ Pro program, at wavelengths 544 (Excitation)and 590 (Emmission). Results confirmed the dose-dependent proliferativeresponse of the BaF3/CRF2-4/zcytor11 cells to IL-TIF-CEE. The response,as measured, was approximately 15-fold over background at the high endof 50 ng/ml down to a 2-fold induction at the low end of 0.06 ng/ml. TheBaF3 wild type cells, and BaF3/CRF2-4 cells did not proliferate inresponse to IL-TIF-CEE, showing that IL-TIF is specific for theCRF24/zcytor11heterodimeric receptor.

In order to determine if Zcytor16 is capable of antagonizing IL-TIFactivity, the assay described above was repeated using purified solublehuman Zcytor16/Fc4. When IL-TIF was combined with Zcytor16 at 10 μg/ml,the response to IL-TIF at all concentrations was brought down tobackground. That the presence of soluble human Zcytor16 ablated theproliferative effects of IL-TIF demonstrates that it is a potentantagonist of the IL-TIF ligand. This assay is also used to assess mouseZcytor16 activity in antagonizing IL-TIF. Similar results are expected.

Example 16 IL-TIF Activation of a Reporter Mini-Gene in MES 13 Cells andInhibition of Activity by Human Zcytor16-Fc4

MES 13 cells (ATCC No. CRL-1927) were plated at 10,000 cells/well in96-well tissue culture clusters (Costar) in DMEM growth medium (LifeTechnologies) supplemented with pyruvate and 10% serum (HyClone). Nextday, the medium was switched to serum free DMEM medium by substituting0.1% BSA (Fraction V; Sigma) for serum. This medium also contained theadenoviral construct KZ136 (below) that encodes a luciferase reportermini-gene driven by SRE and STAT elements, at a 1000:1 multiplicity ofinfection (m.o.i.), i.e. 1000 adenoviral particles per cell. Afterallowing 24 h for the incorporation of the adenoviral construct in thecells, the media were changed and replaced with serum-free media. Humanrecombinant IL-TIF with or without a recombinant Zcytor16-Fc4 fusion wasadded at the indicated final concentration in the well (as described inTable 11, below). Dilutions of both the IL-TIF and Zcytor16-Fc4 wereperformed in serum-free medium. 0.1% BSA was added for a basal assaycontrol. 4 h later, cells were lysed and luciferase activity, denotingactivation of the reporter gene, was determined in the lysate using anLuciferase Assay System assay kit (Promega) and a Labsystems Luminoskanluminometer (Labsystems, Helsinki, Finland). Activity was expressed asluciferase units (LU) in the lysate. Results are shown in Table 11,below. TABLE 11 Level of LU w/o LU w/10 μg/ml IL-TIF (ng/ml) zcytoR16zcytoR16 0 103 ± 2 104 ± 2 (basal BSA control) 0.03 105 ± 3 104 ± 4 0.3108 ± 4  99 ± 6 3 134 ± 8  98 ± 15 30  188 ± 16 110 ± 3 300  258 ± 21 112 ± 30

These results demonstrate two things: First, that MES 13 cells respondto human recombinant IL-TIF and therefore possess endogenous functionalreceptors for the cytokine. Second, that the human zcytoR16-Fc4 receptorfusion acts as an antagonist that effectively blocks the response toIL-TIF, even at the highest dose that this cytokine was used. Therefore,zcyto16 is an effective antagonist of IL-TIF on cells (MES 13) that areintrinsically capable of responding to IL-TIF, i.e. cells that do notrequire exogenous expression of additional receptor components torespond to the cytokine. This assay is also used to assess mouseZcytor16 activity in antagonizing IL-TIF. Similar results are expected.

The construction of the adenoviral KZ136 vector was as follows. Theoriginal KZ136 vector is disclosed in Poulsen, L K et al. J. Biol. Chem.273:6228-6232, 1998. The CMV promoter/enhancer and SV40 pA sequenceswere removed from pACCMV.pLpA (T. C. Becker et al., Meth. Immunology43:161-189, 1994.) and replaced with a linker containing Asp718/KpnI andHindIII sites (oligos ZC13252 (SEQ ID NO:26) and ZC13453 (SEQ IDNO:27)). The STAT/SRE driven luciferase reporter cassette was exisedfrom vector KZ136 (Poulsen, L K et al., supra.).) as aAsp718/KpnI-HindIII fragment and inserted into the adapted pAC vector.Recombinant KZ136 Adenovirus was produced by transfection with JM17Adenovirus into 293 cells as described in T. C. Becker et al. supra.).Plaque purified virus was amplified and used to infect cultured cells at5-50 pfu/cell 12-48 hours before assay. Luciferase reporter assays wereperformed as described in 96 well microplates as per Poulsen, L K etal., supra.).

Example 17 Construct for Generating CEE-Tagged IL-TIF

Oligonucleotides were designed to generate a PCR fragment containing theKozak sequence and the coding region. for human IL-TIF, without its stopcodon. These oligonucleotides were designed with a KpnI site at the 5′end and a BamHI site at the 3′ end to facilitate cloning intopHZ200-CEE, our standard vector for mammalian expression of C-terminalGlu-Glu tagged (SEQ ID NO:10) proteins. The pHZ200 vector contains anMT-1 promoter.

PCR reactions were carried out using Turbo Pfu polymerase (Stratagene)to amplify a IL-TIF cDNA fragment. About 20 ng human IL-TIFpolynucleotide template (SEQ ID NO:14), and oligonucleotides ZC28590(SEQ ID NO:28) and ZC28580 (SEQ ID NO:29) were used in the PCR reaction.PCR reaction conditions were as follows: 95° C. for 5 minutes,; 30cycles of 95° C. for 60 seconds, 55° C. for 60 seconds, and 72° C. for60 seconds; and 72° C. for 10 minutes; followed by a 4° C. hold. PCRproducts were separated by agarose gel electrophoresis and purifiedusing a QiaQuick™ (Qiagen) gel extraction kit. The isolated,approximately 600 bp, DNA fragment was digested with KpnI and BamHI(Boerhinger-Mannheim), gel purified as above and ligated into pHZ200-CEEthat was previously digested with KpnI and BamHI.

About one microliter of the ligation reaction was electroporated intoDH10B ElectroMax™ competent cells (GIBCO BRL, Gaithersburg, Md.)according to manufacturer's direction and plated onto LB platescontaining 100 μg/ml ampicillin, and incubated overnight. Colonies werepicked and screened by PCR using oligonucleotides ZC28,590 (SEQ IDNO:28) and ZC28,580 (SEQ ID NO:29), with PCR conditions as describedabove. Clones containing inserts were then sequenced to confirmerror-free IL-TIF inserts. Maxipreps of the correct pHZ200-IL-TEF-CEEconstruct, as verified by sequence analysis, were performed.

Example 18 Transfection and Expression of IL-TIF Soluble ReceptorPolypeptides

BHK 570 cells (ATCC No. CRL-10314), were plated at about 1.2×10⁶cells/well (6-well plate) in 800 μl of serum free (SF) DMEM media (DMEM,Gibco/BRL High Glucose) (Gibco BRL, Gaithersburg, Md.). The cells weretransfected with an expression plasmid containing IL-TIF-CEE describedabove (Example 17), using Lipofectin™ (Gibco BRL), in serum free (SF)DMEM according to manufacturer's instructions.

The cells were incubated at 37° C. for approximately five hours, thentransferred to separate 150 mm MAXI plates in a final volume of 30 mlDMEM/5% fetal bovine serum (FBS) (Hyclone, Logan, Utah). The plates wereincubated at 37° C., 5% CO₂, overnight and the DNA: Lipofectin™ mixturewas replaced with selection media (5% FBS/DMEM with 1 μM methotrexate(MTX)) the next day.

Approximately 10-12 days post-transfection, colonies were mechanicallypicked to 12-well plates in one ml of 5% FCS/DMEM with 5 μM MTX, thengrown to confluence. Positive expressing clonal colonies Conditionedmedia samples were then tested for expression levels via SDS-PAGE andWestern analysis. A high-expressing clone was picked and expanded forample generation of conditioned media for purification of the IL-TIF-CEEexpressed by the cells (Example 19).

Example 19 Purification of IL-TIF Soluble Receptors from BHK 570 Cells

Unless otherwise noted, all operations were carried out at 4° C. Thefollowing procedure was used for purifying IL-TIF polypeptide containingC-terminal GluGlu (EE) tags (SEQ ID NO: 10). Conditioned media from BHKcells expressing IL-TIF-CEE (Example 18) was concentrated with an AmiconS10Y3 spiral cartridge on a ProFlux A30. A Protease inhibitor solutionwas added to the concentrated conditioned media to final concentrationsof 2.5 mM ethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St.Louis, Mo.), 0.003 mM leupeptin (Boehringer-Mannheim, Indianapolis,Ind.), 0.001 mM pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc(Boehringer-Mannheim). Samples were removed for analysis and the bulkvolume was frozen at −80° C. until the purification was started. Totaltarget protein concentrations of the concentrated conditioned media weredetermined via SDS-PAGE and Western blot analysis with the anti-EE HRPconjugated antibody.

About 10 ml column of anti-EE G-Sepharose (prepared as described below)was poured in a Waters AP-5, 5 cm×10 cm glass column. The column wasflow packed and equilibrated on a BioCad Sprint (PerSeptive BioSystems,Framingham, Mass.) with phosphate buffered saline (PBS) pH 7.4. Theconcentrated conditioned media was thawed, 0.2 micron sterile filtered,pH adjusted to 7.4, then loaded on the column overnight with about 1ml/minute flow rate. The column was washed with 10 column volumes (CVs)of phosphate buffered saline (PBS, pH 7.4), then plug eluted with 200 mlof PBS (pH 6.0) containing 0.5 mg/ml EE peptide (Anaspec, San Jose,Calif.) at 5 ml/minute. The EE peptide used has the sequence EYMPME (SEQID NO:10). The column was washed for 10 CVs with PBS, then eluted with 5CVs of 0.2M glycine, pH 3.0. The pH of the glycine-eluted column wasadjusted to 7.0 with 2 CVs of 5×PBS, then equilibrated in PBS (pH 7.4).Five ml fractions were collected over the entire elution chromatographyand absorbance at 280 and 215 nM were monitored; the pass through andwash pools were also saved and analyzed. The EE-polypeptide elution peakfractions were analyzed for the target protein via SDS-PAGE Silverstaining and Western Blotting with the anti-EE HRP conjugated antibody.The polypeptide elution fractions of interest were pooled andconcentrated from 60 ml to 5.0 ml using a 10,000 Dalton molecular weightcutoff membrane spin concentrator (Millipore, Bedford, Mass.) accordingto the manufacturer's instructions.

To separate IL-TIF-CEE from other co-purifying proteins, theconcentrated polypeptide elution pooled fractions were subjected to aPOROS HQ-50 (strong anion exchange resin from PerSeptive BioSystems,Framingham, Mass.) at pH 8.0. A 1.0×6.0 cm column was poured and flowpacked on a BioCad Sprint. The column was counter ion charged thenequibrated in 20 mM TRIS pH 8.0 (Tris (Hydroxymethyl Aminomethane)). Thesample was diluted 1:13 (to reduce the ionic strength of PBS) thenloaded on the Poros HQ column at 5 ml/minute. The column was washed for10 CVs with 20 mM Tris pH 8.0 then eluted with a 40 CV gradient of 20 mMTris/1 M sodium chloride (NaCl) at 10 ml/minute. 1.5 ml fractions werecollected over the entire chromatography and absorbance at 280 and 215nM were monitored. The elution peak fractions were analyzed via SDS-PAGESilver staining. Fractions of interest were pooled, and concentrated to1.5-2 ml using a 10,000. Dalton molecular weight cutoff membrane spinconcentrator (Millipore, Bedford, Mass.), according to themanufacturer's instructions.

To separate IL-TIF-CEE polypeptide from free EE peptide and anycontaminating co-purifying proteins, the pooled concentrated fractionswere subjected to size exclusion chromatography on a 1.5×90 cm SephadexS200 (Pharmacia, Piscataway, N.J.) column equilibrated and loaded in PBSat a flow rate of 1.0 ml/min using a BioCad Sprint. 1.5 ml fractionswere collected across the entire chromatography and the absorbance at280 and 215 nM were monitored. The peak fractions were characterized viaSDS-PAGE Silver staining, and only the most pure fractions were pooled.This material represented purified IL-TIF-CEE polypeptide.

This purified material was finally subjected to a 4 ml ActiClean Etox(Sterogene) column to remove any remaining endotoxins. The sample waspassed over the PBS equilibrated gravity column four times then thecolumn was washed with a single 3 ml volume of PBS, which was pooledwith the “cleaned” sample. The material was then 0.2 micron sterilefiltered and stored at −80° C. until it was aliquoted.

On Western blotted, Coomassie Blue and Silver stained SDS-PAGE gels, theIL-TIF-CEE polypeptide was one major band. The protein concentration ofthe purified material was performed by BCA analysis (Pierce, Rockford,Ill.) and the protein was aliquoted, and stored at −80° C. according tostandard procedures.

To prepare anti-EE Sepharose, a 100 ml bed volume of protein G-Sepharose(Pharmacia, Piscataway, N.J.) was washed 3 times with 100 ml of PBScontaining 0.02% sodium azide using a 500 ml Nalgene 0.45 micron filterunit. The gel was washed with 6.0 volumes of 200 mM triethanolamine, pH8.2 (TEA, Sigma, St. Louis, Mo.), and an equal volume of EE antibodysolution containing 900 mg of antibody was added. After an overnightincubation at 4° C., unbound antibody was removed by washing the resinwith 5 volumes of 200 mM TEA as described above. The resin wasresuspended in 2 volumes of TEA, transferred to a suitable container,and dimethylpimilimidate-2HCl (Pierce, Rockford, Ill.) dissolved in TEA,was added to a final concentration of 36 mg/ml of protein G-Sepharosegel. The gel was rocked at room temperature for 45 min and the liquidwas removed using the filter unit as described above. Nonspecific siteson the gel were then blocked by incubating for 10 min. at roomtemperature with 5 volumes of 20 mM ethanolamine in 200 mM TEA. The gelwas then washed with 5 volumes of PBS containing 0.02% sodium azide andstored in this solution at 4° C.

Example 20 Human zcytor11 Tissue Distribution in Tissue Panels UsingNorthern Blot and PCR

A. Human zcytor11 Tissue Distribution in Tissue Panels Using PCR

A panel of cDNAs from human tissues was screened for zcytor11 expressionusing PCR. The panel was made in-house and contained 94 marathon cDNAand cDNA samples from various normal and cancerous human tissues andcell lines are shown in Table 6 above. Aside from the PCR reaction, themethod used was as shown in Example12. The PCR reactions were set upusing oligos ZC14,666 (SEQ ID NO: 32) and ZC14,742 (SEQ ID NO:33),Advantage 2 cDNA polymerase mix (Clontech, Palo Alto, Calif.), andRediload dye (Research Genetics, Inc., Huntsville, Ala.). Theamplification was carried out as follows: 1 cycle at 94° C. for 2minutes, 40 cycles of 94° C. for 15 seconds, 51° C. for 30 seconds and72° C. for 30 seconds, followed by 1 cycle at 72° C. for 7 minutes. Thecorrect predicted DNA fragment size was observed in bladder, brain,cervix, colon, fetal brain, fetal heart, fetal kidney, fetal liver,fetal lung, fetal skin, heart, kidney, liver, lung, melanoma, ovary,pancreas, placenta, prostate, rectum, salivary gland, small intestine,testis, thymus, trachea, spinal cord, thyroid, lung tumor, ovariantumor, rectal tumor, and stomach tumor. Zcytor11 expression was notobserved in the other tissues and cell lines tested in this panel.

A commercial 1st strand cDNA panel (Human Blood Fractions MTC Panel,Clontech, Palo Alto, Calif.) was also assayed as above. The panelcontained the following samples: mononuclear cells, activatedmononuclear cells, resting CD4+ cells, activated CD4+ cells, restingCD8+ cells, activated CD8+ cells, resting CD14+ cells, resting CD19+cells and activated CD19+ cells. All samples except activated CD8+ andActivated CD19+ showed expression of zcytor11.

B. Tissue Distribution of Zcytor11 in Human Cell Line and Tissue PanelsUsing RT-PCR

A panel of RNAs from human cell lines was screened for zcytor11expression using RT-PCR. The panels were made in house and contained 84RNAs from various normal and cancerous human tissues and cell lines asshown in Tables 7-10 above. The RNAs were made from in house orpurchased tissues and cell lines using the RNAeasy Midi or Mini Kit(Qiagen, Valencia, Calif.). The panel was set up in a 96-well formatwith 100 ngs of RNA per sample. The RT-PCR reactions were set up usingoligos ZC14,666 (SEQ ID NO:32) and ZC14,742 (SEQ ID NO:33), Rediload dyeand SUPERSCRIPT One Step RT-PCR System(Life Technologies, Gaithersburg,Md.). The amplification was carried out as follows: one cycle at 50° for30 minutes followed by 45 cycles of 94°, 15 seconds; 52°, 30 seconds;72°, 30 seconds; then ended with a final extension at 72° for 7 minutes.8 to 10 uls of the PCR reaction product was subjected to standardAgarose gel electrophoresis using a 4% agarose gel. The correctpredicted cDNA fragment size was observed in adrenal gland, bladder,breast, bronchus, normal colon, colon cancer, duodenum, endometrium,esophagus, gastic cancer, gastro-esophageal cancer, heart ventricle,ileum, normal kidney, kidney cancer, liver, lung, lymph node, pancreas,parotid, skin, small bowel, stomach, thyroid, and uterus. Cell linesshowing expression of zcytor11 were A-431, differentiated CaCO2, DLD-1,HBL-100, HCT-15, HepG2, HepG2+IL6, HuH7, and NHEK #1-4. Zcytor11expression was not observed in the other tissues and cell lines testedin this panel.

In addition, because the expression pattern of zcytor11, one of IL-TIF'sreceptors, shows expression in certain specific tissues, bindingpartners including the natural ligand, IL-TIF, can also be used as adiagnostic to detect specific tissues (normal or abnormal), cancer, orcancer tissue in a biopsy, tissue, or histologic sample, particularly intissues where IL-TIF receptors are expressed. IL-TIF can also be used totarget other tissues wherein its receptors, e.g., Zcytor16 and zcytor11are expressed. Moreover, such binding partners could be conjugated tochemotherapeutic agents, toxic moieties and the like to target therapyto the site of a tumor or diseased tissue. Such diagnostic and targetedtherapy uses are known in the art and described herein.

The expression patterns of zcytor11 (above) and Zcytor16 (Example 12,and Example 21) indicated target tissues and cell types for the actionof IL-TIF, and hence IL-TIF antagonsists, such as Zcytor16. The zcytor11expression generally overlapped with Zcytor16 expression in threephysiologic; systems: digestive system, female reproductive system, andimmune system. Moreover, the expression pattern of the receptor(zcytor11) indicated that an IL-TIF antagonist such as Zcytor16 wouldhave therapeutic application for human disease in two areas:inflammation (e.g., IBD, Chron's disease, pancreatitis) and cancer(e.g., ovary, colon). That is, the polynucleotides, polypeptides andantibodies of the present invention can be used to antagonize theinflammatory, and other cytokine-induced effects of IL-TIF interactionwith the cells expressing the zcytor11 receptor.

Moreover, the expression of zcytor11 appeared to be downregulated orabsent in an ulcerative colitis tissue, HepG2 liver cell line induced byIL-6, activated CD8+ T-cells and CD19+ B-cells. However, Zcytor16appeared to be upregulated in activated CD19+ B-cells (Example 12),while zcytor11 is downregulated in activated CD19+ cells, as compared tothe resting CD19+ cells (above). The expression of zcytor11 and Zcytor16has a reciprocal correlation in this case. These RT-PCR experimentsdemonstrate that CD19+ peripheral blood cells, B lymphocytes, expressreceptors for IL-TIF, namely zcytoR11 and zcytoR16. Furthermore B cellsdisplay regulated expression of zcytoR11 and zcytoR16. B-lymphocytesactivated with mitogens decrease expression of zcytoR11 and increaseexpression of zcytoR16. This represents a classical feedback inhibitionthat would serve to dampen the activity of IL-TIF on B cells and othercells as well. Soluble zcytoR16 would act as an antagonist to neutralizethe effects of IL-TIF on B cells. This would be beneficial in diseaseswhere B cells are the key players: Autoimmune diseases includingsystemic lupus erythmatosus (SLE), myasthenia gravis, immune complexdisease, and B-cell cancers that are exacerbated by IL-TIF. Alsoautoimmune diseases where B cells contribute to the disease pathologywould be targets for zcytoR16 therapy: Multiple sclerosis, inflammatorybowel disease (IBD) and rheumatoid arthritis are examples. ZcytoR16therapy would be beneficial to dampen or inhibit B cells producing IgEin atopic diseases including asthma, allergy and atopic dermatitis wherethe production of IgE contributes to the pathogenesis of disease.

B cell malignancies may exhibit a loss of the “feedback inhibition”described above. Administration of zcytoR16 would restore control ofIL-TIF signaling and inhibit B cell tumor growth. The administration ofzcytoR16, following surgical resection or chemotherapy may be useful totreat minimal residual disease in patients with B cell malignancies. Theloss of regulation may lead to sustain or increased expression ofzcytoR11. Thus creating a target for therapeutic monoclonal antibodiestargeting zcytoR11.

Example 21 Identification of Cells Expressing Zcytor16 Using in SituHybridization

Specific human tissues were isolated and screened for human Zcytor16expression by in situ hybridization. Various human tissues prepared,sectioned and subjected to in situ hybridization included cartilage,colon, appendix, intestine, fetal liver, lung, lymph node, lymphoma,ovary, pancreas, placenta, prostate, skin, spleen, and thymus. Thetissues were fixed in 10% buffered formalin and blocked in paraffinusing standard techniques. Tissues were sectioned at 4 to 8 microns.Tissues were prepared using a standard protocol (“Development ofnon-isotopic in situ hybridization” at The Laboratory of ExperimentalPathology (LEP), NIEHS, Research Triangle Park, N.C.; web addresshttp://dir.niehs.nih.gov/dirlep/ish.html). Briefly, tissue sections weredeparaffinized with HistoClear (National Diagnostics, Atlanta, Ga.) andthen dehydrated with ethanol. Next they were digested with Proteinase K(50 μg/ml) (Boehringer Diagnostics, Indianapolis, Ind.) at 37° C. for 2to 7 minutes. This step was followed by acetylation and re-hydration ofthe tissues.

One in situ probe was designed against the human Zcytor16 sequence(nucleotide 1-693 of SEQ ID NO:1), and isolated from a plasmidcontaining SEQ ID NO:1 using standard methods. T3 RNA polymerase wasused to generate an antisense probe. The probe was labeled withdigoxigenin (Boehringer) using an In Vitro transcription System(Promega, Madison, Wis.) as per manufacturer's instruction.

In situ hybridization was performed with a digoxigenin-labeled Zcytor16probe (above). The probe was added to the slides at a concentration of 1to 5 pmol/ml for 12 to 16 hours at 62.5° C. Slides were subsequentlywashed in 2×SSC and 0.1×SSC at 55° C. The signals were amplified usingtyramide signal amplification (TSA) (TSA, in situ indirect kit; NEN) andvisualized with Vector Red substrate kit (Vector Lab) as permanufacturer's instructions. The slides were then counter-stained withhermatoxylin (Vector Laboratories, Burlingame, Calif.).

Signals were observed in several tissues tested: The lymph node, plasmacells and other mononuclear cells in peripheral tissues were stronglypositive. Most cells in the lymphatic nodule were negative. In lymphomasamples, positive signals were seen in the mitotic and multinuclearcells. In spleen, positive signals were seen in scattered mononuclearcells at the periphery of follicles were positive. In thymus, positivesignals were seen in scattered mononuclear cells in both cortex andmedulla were positive. In fetal liver, a strong signal was observed in amixed population of mononuclear cells in sinusoid spaces. A subset ofhepatocytes might also have been positive. In the inflamed appendix,mononuclear cells in peyer's patch and infiltration sites were positive.In intestine, some plasma cells and ganglia nerve cells were positive.In normal lung, Zcytor16 was expressed in alveolar epithelium andmononuclear cells in interstitial tissue and circulation. In the lungcarcinoma tissue, a strong signal was observed in mostly plasma cellsand some other mononuclear cells in peripheral of lymphatic aggregates.In ovary carcinoma, epithelium cells were strongly positive. Someinterstitial cells, most likely the mononuclear cells, were alsopositive. There was no signal observed in the normal ovary. In bothnormal and pancreatitis pancreas samples, acinar cells and somemononuclear cells in the mesentery were positive. In the early term (8weeks) placenta, signal was observed in trophoblasts. In skin, somemononuclear cells in the inflamed infiltrates in the superficial dermiswere positive. Keratinocytes were also weakly positive. In prostatecarcinoma, scatted mononuclear cells in interstitial tissues werepositive. In articular cartilage, chondrocytes were positive. Othertissues tested including normal ovary and a colon adenocarcinoma werenegative.

In summary, the in situ data was consistent with expression datadescribed above for the Zcytor16. Zcytor16 expression was observedpredominately in mononuclear cells, and a subset of epithelium was alsopositive. These results confirmed the presence of Zcytor16 expression inimmune cells and point toward a role in inflammation, autoimmunedisease, or other immune function, for example, in bindingpro-inflammatory cytokines, including but not limited to IL-TIF.Moreover, detection, of Zcytor16 expression can be used for, example asan marker for mononuclear cells in histologic samples.

Zcytor16 is expressed in mononuclear cells, including normal tissues(lymph nodes, spleen, thymus, pancreas and fetal liver, lung), andabnormal tissues (inflamed appendix, lung carcinoma, ovary carcinoma,pancreatitis, inflamed skin, and prostate carcinoma). It is notable thatplasma cells in the lymph node, intestine, and lung carcinoma arepositive for Zcytor16. Plasma cells are immunologically activatedlymphocytes responsible for antibody synthesis. In addition, IL-TIF, isexpressed in activated T cells. In addition, the expression of Zcytor16is detected only in activated (but not in resting) CD4+ and CD19+ cells(Example 12). Thus, Zcytor16 can be used as a marker for or as a targetin isolating certain lymphocytes, such as mononuclear leucocytes andlimited type of activated leucocytes, such as activated CD4+ and CD19+.

Furthermore, the presence of Zcytor16 expression in activated immunecells such as activated CD4+ and CD19+ cells showed that Zcytor16 may beinvolved in the body's immune defensive reactions against foreigninvaders: such as microorganisms and cell debris, and could play a rolein immune responses during inflammation and cancer formation.

Moreover, as discussed herein, epithelium form several tissues waspositive for Zcytor16 expression, such as hepatocytes (endoderm-derivedepithelia), lung alveolar epithelium (endoderm-derived epithelia), andovary carcinoma epithelium (mesoderm-derived epithelium). The epitheliumexpression of Zcytor16 could be altered in inflammatory responses and/orcancerous states in liver and lung. Thus, Zcyto16 could be used asmarker to monitor changes in these tissues as a result of inflammationor cancer. Moreover, analysis of Zcytor16 in situ expression showed thatnormal ovary epithelium is negative for Zcytor16 expression, while it isstrongly positive in ovary carcinoma epithelium providing furtherevidence that Zcytor16 polynucleotides, polypeptides and antibodies canbe used as a diagnostic marker and/or therapeutic target for thediagnosis and treatment of ovarian cancers, and ovary carcinoma, asdescribed herein.

Zcytor16 was also detected in other tissues, such as acinar cells inpancreas (normal and pancreatitis tissues), trophoblasts in placenta(ectoderm-derived); chondrocytes in cartilage (mesoderm-derived), andganglia cells in intestine (ectoderm-derived). As such, Zcytor16 may beinvolved in differentiation and/or normal functions of correspondingcells in these organs. As such, potential utilities of Zcytor16 includemaintenance of normal metabolism and pregnancy, boneformation/homeostasis, and physiological function of intestine, and thelike.

Example 22 In Vivo Effects of IL-TIF Polypeptide

Mice (female, C57B1, 8 weeks old; Charles River Labs, Kingston, N.Y.)were divided into three groups. An adenovirus expressing an IL-TIFpolypeptide (SEQ ID NO:15) was previously made using standard methods.On day 0, parental or IL-TIF adenovirus was administered to the first(n=8) and second (n=8) groups, respectively, via the tail vein, witheach mouse receiving a dose of ˜1×10¹¹ particles in ˜0.1 ml volume. Thethird group (n=8) received no treatment. On days 12, mice were weighedand blood was drawn from the mice. Samples were analyzed for completeblood count (CBC) and serum chemistry. Statistically significantelevations in neutrophil and platelet counts were detected in the bloodsamples from the IL-TIF adenovirus administered group relative to theparental adenovirus treated group. Also, lymphocyte and red blood cellcounts were significantly reduced from the IL-TIF adenovirusadministered group relative to the parental adenovirus treated group. Inaddition, the IL-TIF adenovirus treated mice decreased in body weight,while parental adenovirus treated mice gained weight.

The results suggested that IL-TIF affects hematopoiesis, i.e., bloodcell formation in vivo. As such, IL-TIF could have biological activitieseffecting different blood stem cells, thus resulting increase ordecrease of certain differentiated blood cells in a specific lineage.For instance, IL-TIF appears to reduce lymphocytes, which is likely dueto inhibition of the committed progenitor cells that give rise tolymphoid cells. IL-TIF also decreases red blood cells. This findingagrees with the inhibitory effects of IL-TIF on the proliferation and/orgrowth of myeloid stem cells (Example 23), supporting the notion thatIL-TIF could play a role in anemia, infection, inflammation, and/orimmune diseases by influencing blood cells involved in these processes.Antagnists against IL-TIF, such as antibodies or its soluble receptorZcytor16, could be used as therapeutic reagents in these diseases.

Moreover, these experiments using IL-TIF adenovirus in mice suggest thatIL-TIF over-expression increases the level of neutrophils and plateletsin vivo. Although this may appear contradictory to the finding seen inK562 cells (Example 23), it is not uncommon to observe diverseactivities of a particular protein in vitro versus in vivo. It isconceivable that there are other factors (such as cytokines and modifiergenes) involved in the responses to IL-TIF in the whole animal system.Nevertheless, these data strongly support the involvement of IL-TIF inhematopoiesis. Thus, IL-TIF and its receptors are suitablereagents/targets for the diagnosis and treatment in variety ofdisorders, such as inflammation, immune disorders, infection, anemia,hematopoietic and other cancers, and the like.

Example 23 The IL-TIF Polypeptide Lyses K-562 Cells in CytotoxicityAssay

The K-562 cell line (CRL-243, ATCC) has attained widespread use as ahighly sensitive in vitro target for cytotoxicity assays. K-562 blastsare multipotential, hematopoietic malignant cells that spontaneouslydifferentiate into recognizable progenitors of the erythrocytic,granulocytic and monocytic series (Lozzio, B B et al., Proc. Soc. Exp.Biol. Med. 166: 546-550, 1981).

K562 cells were plated at 5,000 cells/well in 96-well tissue cultureclusters (Costar) in DMEM phenol-free growth medium (Life Technologies)supplemented with pyruvate and 10% serum (HyClone). Next day, humanrecombinant IL-TIF (Example 19), BSA control or retinoic acid (known tobe cytotoxic to K562 cells) were added. Seventy-two hours later, thevital stain MTT (Sigma, St Louis, Mo.), a widely used indicator ofmitochondrial activity and cell growth, was added to the cells at afinal concentration of 0.5 mg/ml. MMP is converted to a purple formazanderivative by mitochondrial dehydrogenases. Four hours later, convertedMMP was solubilized by adding an equal volume of acidic, isopropanol(0.04N HCl in absolute isopropanol) to the wells. Absorbance wasmeasured at 570 nm, with background subtraction at 650 nm. In thisexperimental setting, absorbance reflects cell viability. Results shownin Table 12 are expressed as % cytotoxicity. TABLE 12 AgentConcentration % Cytotoxicity BSA Control 1 ug/ml 1.3 Retinoic acid 100uM 62 IL-TIF 100 ng/ml 16.2 IL-TIF 300 ng/ml 32

The results indicate that IL-TIF may affect myeloid stem cells. Myeloidstem cells are daughter cells of the universal blood stem cells. Theyare progenitors of erythrocytes, platelets megakaryocytes, monocytes (ormigrated macrophages), neutrophil and basophil, etc. Since K-562 blastsspontaneously differentiate into progenitors of the erythrocytic,granulocytic and monocytic series, it can be considered as myeloid stemcells. Thus, the results demonstrate that IL-TIF has an inhibitoryactivity on the proliferation and/or growth of myeloid stem cells. ThusIL-TIF could play a role in anemia, infection, inflammation, and/orimmune diseases. In addition, an antaganist against IL-TIF, such asantibodies or its soluble receptor Zcytor16, could be used to blockIL-TIF's activity on myeloid stem cells, or as therapeutic reagents indiseases such as anemia, infection, inflammation, and/or immunediseases.

Example 24 IL-TIF-Expressing Transgenic Mice

A. Generation of Transgenic Mice Expressing Mouse IL-TIF

DNA fragments from a transgenic vector containing 5′ and 3′ flankingsequences of the lymphoid specific EμLCK promoter, mouse IL-TIF (SEQ IDNO:40), the rat insulin II intron, IL-TIF cDNA and the human growthhormone poly A sequence were prepared using standard methods, and usedfor microinjection into fertilized B6C3f1 (Taconic, Germantown, N.Y.)murine oocytes, using a standard microinjection protocol. See, Hogan, B.et al., Manipulating the Mouse Embryo. A Laboratory Manual, Cold SpringHarbor Laboratory Press, 1994.

Twenty-five mice transgenic for mouse IL-TIF with the lymphoid-specificEμLCK promoter were identified among 154 pups. Eleven of the transgenicpups died within hours of birth, 9 transgenic pups with a shinyappearance were necropsied the day of birth, and 2 grew to adulthood.Expression levels were low in one adult animal. Tissues from thenecropsied pups were prepared and histologically examined as describedbelow.

The shiny appearance of the neonate pups appeared to be associated witha stiffening of the skin, as if they were drying out, resulting in areduction of proper nursing. Their movements became stiffened ingeneral.

B. Genotypic and Expression Analysis from Transgenic Mice

From the mouse IL-TIF transgenic line driven by the EμLck promoter,described above, newborn pups were observed for abnormalities on day one(day of birth) and sacrificed for tissue collection. All pups were givena unique ear tag number, and those designated as having a shiny skinphenotype at the time of sacrifice were noted. Of the twelve pups, sixwere observed to have the shiny skin phenotype, with two designated as“severe” phenotypes. Severe phenotypes were defined as small pups withlittle mobility whose skin was especially shiny and very dry. Skin wascollected from the left lateral side of each pup, and frozen inTissue-Tek embedding medium.

Genotyping confirmed that shiny skin was a good indicator of transgenicstatus, although no expression data was collected. Frozen skin blockswere sectioned to 7 microns on a cryostat and stained to look for thepresence of CD3, CD4, CD8, mouse macrophages, B-cells, CD80, and MHCclass II. The staining protocol involved binding of commerciallyavailable antibodies to the tissue, detection with a peroxidase labeledsecondary antibody, and DAB chromogen reaction to visualize staining.

Transgenic animals were found to be higher in MHC class II and CD80,which stain for antigen-presenting cells and dendritic cellsrespectively. The macrophage marker also detected more cells in thesevere and non-severe transgenics than in the wild type animals,although the distribution of these cells was very localized in the highdermis. Animals classified as severe phenotypes had the most robuststaining with all three of these markers, showing a dramatic increase incell intensity and number when compared to the wild type. Thisvariability may be due to a difference in expression level of IL-TIF inthese transgenic founder pups. The MHC class II positive cells werelocated in the lower dermis arranged in loose open clusters, while theCD80 positive cells were predominantly below the dermis either in orjust above the muscle/fat layer. These two cell populations do notappear to overlap. All other markers were of equivalent staining in allanimals. Toluidine blue staining for mast cells revealed slight to nodifference between wild type and transgenic animals.

C. Microscopic Evaluation of Tissues from Transgenic Mice: IL-TIF TGwith EuLck Promoter has a Neonatal Lethal-Histology

On the day of birth, pups from litters containing IL-TIF transgenicswere humanely euthanized and the whole body immersion fixed in 10%buffered formalin. Six transgenic and two non-transgenic pups weresubmitted for further workup. Four of the six transgenics were noted tohave shiny skin at the time of euthanasia. The fixed tissues weretrimmed into 5 sections (longitudinal section of the head and crosssections of the upper and lower thorax and upper and lower abdomen). Thetissues were embedded in paraffin, routinely processed, sectioned at 5um (Jung 2065 Supercut microtome, Leica Microsystems, Wetzlar, Germany)and stained with H&E. The stained tissues were evaluated under a lightmicroscope (Nikon Eclipse E600, Nikon Inc., Melville, N.Y.) by a board(ACVP) certified veterinary pathologist.

On microscopic examination, the epidermis of two of the transgenic pupswas observed to be thicker than the epidermis of the other six miceincluding the controls. No other abnormalities were noted in the skinand other tissues of any of the mice. Representative areas of skin fromcorresponding regions of the thorax and abdomen were imaged with the 40×objective lens and with a CoolSnap digital camera (Roper Scientific,Inc., San Diego, Calif.) that was attached to the microscope. Thethickness of the epidermis was then determined using histomorphometrysoftware (Scion Image for Windows (NIH Image), Scion Corp., Frederick,Md., v. B4.0.2). The results were as follows: Average thoracic skinAverage abdominal skin Genotype/phenotype thickness (μm) thickness (μm)Non-transgenic/normal 5.2 5.4 Transgenic/non-shiny 5.0 6.7Transgenic/shiny 8.2 7.4 Transgenic/all 7.1 7.1

There were insufficient numbers of mice to determine statisticalsignificance; however, the transgenics, especially those with shinyskin, tended to have a thicker epidermis than the non-shiny transgenicsand non-transgenic controls. The shiny transgenics may have a higherexpression level of IL-TIF than the non-shiny transgenics.; however,expression levels were not determined for these mice.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. An isolated polynucleotide comprising a nucleic acid sequence fromthe group consisting of: (a) the polynucleotide sequence from 8 to 697of SEQ ID NO:47; (b) the polynucleotide sequence from 77 to 697 of SEQID NO:47; (c) the polynucleotide sequence from 86 to 697 of SEQ IDNO:47; and (d) polynucleotide sequences complementary to thepolynucleotides encoding the polypeptides consisting of a sequence ofamino acid residues as shown in (a), (b), and (c).
 2. An isolatedpolynucleotide of claim 1, wherein the polynucleotide consists of anucleic acid sequence from the group consisting of: (a) thepolynucleotide sequence from 8 to 697 of SEQ ID NO:47; (b) thepolynucleotide sequence from 77 to 697 of SEQ ID NO:47; (c) thepolynucleotide sequence from 86 to 697 of SEQ ID NO:47; and (d)polynucleotide sequences complementary to the polynucleotides encodingthe polypeptides consisting of a sequence of amino acid residues asshown in (a), (b), and (c).
 3. An isolated polynucleotide encoding apolypeptide comprising a sequence of amino acid residues selected fromthe group consisting of: (a) amino acid residues 24 to 230 of SEQ IDNO:48; (b) amino acid residues 27 to 230 of SEQ ID NO:48; (c) amino acidresidues 27 to 126 of SEQ ID NO:48; (d) amino acid residues 131 to 230of SEQ ID NO:48; and (e) amino acid residues 1 to 230 of SEQ ID NO:48.4. An isolated polynucleotide of claim 3, wherein the polynucleotideencodes a polypeptide consisting of a sequence of amino acid residuesselected from the group consisting of: (a) amino acid residues 24 to 230of SEQ ID NO:48; (b) amino acid residues 27 to 230 of SEQ ID NO:48; (c)amino acid residues 27 to 126 of SEQ ID NO:48; (d) amino acid residues131 to 230 of SEQ ID NO:48; and (e) amino acid residues 1 to 230 of SEQID NO:48.
 5. The isolated polynucleotide of claim 1, comprising thenucleotide sequence of nucleotides 8 to 697 of SEQ ID NO:47, or 77 to697 of SEQ ID NO:47, or 86 to 697 of SEQ ID NO:47.
 6. A vector,comprising the isolated polynucleotide of claim
 3. 7. An expressionvector, comprising the isolated polynucleotide of claim 3, atranscription promoter, and a transcription terminator, wherein thepromoter is operably linked with the polynucleotide, and wherein thepolynucleotide is operably linked with the transcription terminator. 8.A recombinant host cell comprising the expression vector of claim 7,wherein the host cell is selected from the group consisting ofbacterium, yeast cell, fungal cell, insect cell, mammalian cell, andplant cell.
 9. A method of producing mouse Zcytor16 protein, the methodcomprising culturing recombinant host cells that comprise the expressionvector of claim 7, and that produce the mouse Zcytor16 protein.
 10. Themethod of claim 9, further comprising isolating the mouse Zcytor16protein from the cultured recombinant host cells.
 11. An isolatedpolynucleotide that encodes a soluble cytokine receptor polypeptidecomprising a sequence of amino acid residues from amino acid 24 to 230of SEQ ID NO:48, or 27 to 230 of SEQ ID NO:48; and wherein the solublecytokine receptor polypeptide encoded by the polynucleotide sequencebinds IL-10-Related T Cell-Derived Inducible Factor (IL-TIF) orantagonizes IL-TIF activity.
 12. An isolated polynucleotide according toclaim 11, wherein the soluble cytokine receptor polypeptide encoded bythe polynucleotide comprises a homodimeric, heterodimeric or multimericreceptor complex.
 13. An isolated polynucleotide according to claim 12,wherein the soluble cytokine receptor polypeptide encoded by thepolynucleotide comprises a heterodimeric or multimeric receptor complexfurther comprising a soluble Class I or Class II cytokine receptor. 14.An isolated polynucleotide according to claim 12, wherein the solublecytokine receptor polypeptide encoded by the polynucleotide comprises aheterodimeric or multimeric receptor complex further comprising asoluble Cytokine Receptor Family 2-4 (CRF2-4) receptor polypeptide (SEQID NO:35), a soluble IL-10 receptor polypeptide (SEQ ID NO:36), orsoluble zcytor11 receptor polypeptide (SEQ ID NO:34).
 15. An isolatedpolynucleotide that encodes a soluble cytokine receptor polypeptidecomprising a sequence of amino acid residues as shown from amino acid 24to 230 of SEQ ID NO:48, or 27 to 230 of SEQ ID NO:48, wherein thesoluble cytokine receptor polypeptide encoded by the polynucleotidecomprises a homodimeric, heterodimeric or multimeric receptor complex.16. An isolated polynucleotide according to claim 15, wherein thesoluble cytokine receptor polypeptide encoded by the polynucleotidefurther comprises a soluble Class I or Class II cytokine receptor. 17.An isolated polynucleotide according to claim 15, wherein the solublecytokine receptor polypeptide encoded by the polynucleotide comprises aheterodimeric or multimeric receptor complex further comprising asoluble Cytokine Receptor Family 2-4 (CRF2-4) receptor polypeptide (SEQID NO:35), a soluble IL-10 receptor polypeptide (SEQ ID NO:36), orsoluble zcytor11 receptor polypeptide (SEQ ID NO:34).
 18. An isolatedpolynucleotide according to claim 15, wherein the soluble cytokinereceptor polypeptide further encodes an intracellular domain.
 19. Anisolated polynucleotide according to claim 15, wherein the solublecytokine receptor polypeptide further comprises an affinity tag.
 20. Anexpression vector comprising the following operably linked elements: (a)a transcription promoter; a first DNA segment encoding a solublecytokine receptor polypeptide having an amino acid sequence as shownfrom amino acid 24 to 230 of SEQ ID NO:48, or 27 to 230 of SEQ ID NO:48;and a transcription terminator; and (b) a second transcription promoter;a second DNA segment encoding a soluble Class I or Class II cytokinereceptor polypeptide; and a transcription terminator; and wherein thefirst and second DNA segments are contained within a single expressionvector or are contained within independent expression vectors.
 21. Anexpression vector according to claim 20, further comprising a secretorysignal sequence operably linked to the first and second DNA segments.22. An expression vector according to claim 20, wherein the second DNAsegment encodes a polypeptide comprising a soluble Cytokine ReceptorFamily 2-4 (CRF2-4) receptor polypeptide (SEQ ID NO:35), a soluble IL-10receptor polypeptide (SEQ ID NO:36), or soluble zcytor11 receptorpolypeptide (SEQ ID NO:34).
 23. A cultured cell comprising an expressionvector according to claim 20, wherein the cell expresses thepolypeptides encoded by the DNA segments.
 24. A cultured cell comprisingan expression vector according to claim 20, wherein the first and secondDNA segments are located on independent expression vectors and areco-transfected into the cell, and cell expresses the polypeptidesencoded by the DNA segments.
 25. A cultured cell into which has beenintroduced an expression vector according to claim 20, wherein the cellexpresses a heterodimeric or multimeric soluble receptor polypeptideencoded by the DNA segments.
 26. A cell according to claim 23, whereinthe cell secretes a soluble cytokine receptor polypeptide heterodimer ormultimeric complex.
 27. A cell according to claim 23, wherein the cellsecretes a soluble cytokine receptor polypeptide heterodimer ormultimeric complex that binds IL-10-Related T Cell-Derived InducibleFactor (IL-TIF) or antagonizes IL-TIF activity.
 28. A DNA constructencoding a fusion protein comprising: a first DNA segment encoding apolypeptide having a sequence of amino acid residues as shown from aminoacid 24 to 230 of SEQ ID NO:48, or 27 to 230 of SEQ ID NO:48; and atleast one other DNA segment encoding a soluble Class I or Class IIcytokine receptor polypeptide, wherein the first and other DNA segmentsare connected in-frame; and wherein the first and other DNA segmentsencode the fusion protein.
 29. A DNA construct encoding a fusion proteinaccording to claim 28, wherein at least one other DNA segment encodes apolypeptide comprising a soluble Cytokine Receptor Family 2-4 (CRF2-4)receptor polypeptide (SEQ ID NO:35), a soluble EL-10 receptorpolypeptide (SEQ ID NO:36), or soluble zcytor11 receptor polypeptide(SEQ ID NO:34).
 30. An expression vector comprising the followingoperably linked elements: a transcription promoter; a DNA constructencoding a fusion protein according to claim 28; and a transcriptionterminator, wherein the promoter is operably linked to the DNAconstruct, and the DNA construct is operably linked to the transcriptionterminator.
 31. A cultured cell comprising an expression vectoraccording to claim 30, wherein the cell expresses a polypeptide encodedby the DNA construct.
 32. A method of producing a fusion proteincomprising: culturing a cell according to claim 31; and isolating thepolypeptide produced by the cell.
 33. A method of producing a solublecytokine receptor polypeptide that comprises a heterodimeric ormultimeric complex comprising: culturing a cell according to claim 23;and isolating the soluble receptor polypeptides produced by the cell.34. The isolated polynucleotide according to claim 15, wherein thesoluble cytokine receptor polypeptide further comprises an affinity tag,chemical moiety, toxin, label, biotin/avidin label, radionuclide,enzyme, substrate, cofactor, inhibitor, fluorescent marker,chemiluminescent marker, cytotoxic molecule or an immunoglobulin Fcdomain.
 35. The isolated polynucleotide according to claim 3, whereinthe polypeptide by the polynucleotide further comprises an affinity tag,chemical moiety, toxin, label, biotin/avidin label, radionuclide,enzyme, substrate, cofactor, inhibitor, fluorescent marker,chemiluminescent marker, cytotoxic molecule or an immunoglobulin Fcdomain.
 36. The isolated polynucleotide of claim 3, wherein thepolypeptide encoded by the polynucleotide binds IL-10-Related TCell-Derived Inducible Factor (IL-TIF) or antagonizes IL-TIF activity.